Transmembrane protein expressed in prostate and other cancers

ABSTRACT

A novel prostate tumor associated gene (designated 24P4C12) and its encoded protein is described. 24P4C12 is highly expressed in prostate tissue xenografts, providing evidence that it is turned on in at least some prostate cancers. 24P4C12 provides a diagnostic and/or therapeutic target for prostate and other cancers.

This application is a divisional of U.S. Ser. No. 09/547,789, filed 12Apr. 2000, and now U.S. Pat. No. 6,943,235, which application claims thebenefit of U.S. provisional application No. 60/128,858, filed Apr. 12,1999. The contents of these applications are incorporated herein byreference.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed 24P4C12, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that express24P4C12, particularly prostate cancers.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic disease progression, includingthe transition from androgen dependence to androgen independence and thedevelopment of metastatic lesions (Klein et al., 1997, Nat. Med.3:402).More recently identified prostate cancer markers include PCTA-1 (Su etal., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate stem cellantigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735), and STEAP (Hubert et al., 1999, Proc. Natl. Acad. Sci. USA 96:14523).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention relates to a novel family of genes and proteins,characterized by multiple transmembrane regions and expression inprostate cancer. More particularly, the invention provides a novel geneand protein, termed 24P4C12. The 24P4C12 gene encodes a 710 amino acidprotein containing 13 transmembrane domains and bearing homology tomurine and C. elegans genes containing 12 transmembrane domains. Thenucleotide and encoded amino acid sequences of the entire coding andpartial non-coding regions of the human 24P4C12 gene are shown in FIGS.1A–1D (SEQ ID NOS: 1, 2). RT-PCR and Northern blot analyses showexpression of 24P4C12 in normal colon, prostate, kidney and lung, and inprostate cancer xenografts. The transmembrane nature of the 24P4C12protein, combined with its expression in prostate cancer, suggest that24P4C12 is a target for prostate cancer therapy using, for example,antibodies and other small molecules capable of binding to andmodulating the 24P4C12 protein in vivo. In addition, because of itslocation on the cell surface of prostate cancer cells, antibodies andother agents capable of detecting 24P4C12 protein can be useful inprostate cancer imaging methods. Various other molecular detectionassays using, for example, polynucleotide probes and primers capable ofdetecting 24P4C12 transcription products, may also find use indiagnosing, monitoring, prognosing, and staging prostate cancer andpotentially other cancers.

The invention provides polynucleotides corresponding or complementary tothe 24P4C12 gene, mRNA, or fragments thereof, including cDNAs, RNAs,oligonucleotide probes, and primers. The invention further providesmethods for detecting the presence of 24P4C12 polynucleotides in variousbiological samples. Molecular diagnostic assays for prostate cells using24P4C12 polynucleotides are also provided. Such assays may providediagnostic and/or prognostic information concerning the presence anddegree of prostate cancer. The invention further provides means forisolating cDNAs and the gene encoding 24P4C12, as well as those encodingmutated and other forms of 24P4C12. Recombinant DNA molecules containing24P4C12 polynucleotides, cells transformed or transduced with suchmolecules, and host-vector systems for the expression of 24P4C12 geneproducts are also provided. The invention further provides 24P4C12proteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to 24P4C12 proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker.

The invention further provides methods for detecting the presence andstatus of 24P4C12 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 24P4C12.A typical embodiment of this invention provides methods for monitoring24P4C12 gene products in a tissue sample having or suspected of havingsome form of growth disregulation such as cancer.

The invention further provides various therapeutic compositions andstrategies for treating cancers that express 24P4C12 such as cancer ofthe prostate, including therapies aimed at inhibiting the transcription,translation, processing or function of 24P4C12 as well as cancervaccines.

In addition, the invention provides a novel gene and protein related to24P4C12, termed H38087. The H38087 gene encodes a 704 amino acid proteincontaining 11 potential transmembrane domains. The nucleotide andencoded amino acid sequences of the entire coding and partial non-codingregions of the human H38087 gene are shown in FIGS. 7A–7D (SEQ ID NOS:6, 7). The 58 base pairs of 5′ untranslated region are very GC rich(87%), indicating that this gene may contain translational regulatoryelements. The amino acid sequences of 24P4C12 and H38087 are 44%identical and 56% homologous over the entire sequence (FIG. 8).Expression analysis shows that H38087 is ubiquitously expressed (FIG.9), with highest expression levels detected in testis. Expression isalso observed in each of the various LAPC xenografts examined. H38087could serve as a control for testing 24P4C12-specific therapeutics, orprovide a diagnostic and/or therapeutic target. A therapeutic thatselectively affects 24P4C12, but not H38087, may be less toxic to normalcells. Therefore, H38087 protein may be useful as a pre-clinical testingtool for therapeutic modalities directed towards 24P4C12. H38087 proteinexpression, however, may be less ubiquitous than its RNA expression,suggesting H38087 as a target for diagnostic and therapeutic strategies.

The invention additionally provides a method for identifying a 24P4C12specific binding agent. The method comprises contacting a candidateagent that binds 24P4C12 with H38087, and determining whether thecandidate agent binds H38087. A lack of binding of the candidate agentto H38087 being indicative of 24P4C12 specificity. Such binding can bedetected using conventional binding assays known in the art, includingrepresentative assays described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1D. Nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ IDNO: 2) sequences of entire coding region (and part of the 3′ non-codingregion) of the 24P4C12 gene. This sequence was generated from theoverlapping sequences of three cDNA clones, designated 24P4C12-GTE9,24P4C12-GTE5 and 24P4C12-GTE4(Example 2). Thirteen potentialtransmembrane domains are underlined in bold. A Kozak sequence andputative start methionine are indicated in bold.

FIG. 1E. Nucleotide (SEQ ID NO: 3) and ORF amino acid (SEQ ID NO: 4)sequences of the initially isolated SSH fragment of the 24P4C12 gene.

FIG. 2A. RT-PCR analysis of 24P4C12 gene expression in prostate cancerxenografts, normal prostate, and other tissues and cell lines, showingapproximately equal levels of expression in normal prostate and the LAPCprostate cancer xenografts. Lanes represent the following tissues: (1)brain; (2) prostate; (3) LAPC-4 AD; (4) LAPC-4 AI; (5) LAPC-9 AD; (6)HeLa; (7) murine cDNA; and (8) negative control.

FIG. 2B. RT-PCR analysis of 24P4C12 gene expression in various tissues,showing detectable expression only in normal kidney and lung after 25cycles of PCR amplification. Lower level expression is detectable in avariety of other tissues after 30 cycles of amplification. Lanesrepresent the following tissues: (1) brain; (2) heart; (3) kidney; (4)liver; (5) lung; (6) pancreas; (7) placenta; and (8) skeletal muscle.

FIG. 2C. RT-PCR analysis of 24P4C12 gene expression in various tissues,showing detectable expression only in normal colon and prostate after 25cycles of PCR amplification. Lower level expression is detectable in avariety of other tissues after 30 cycles of amplification. Lanesrepresent the following tissues: (1) colon; (2) ovary; (3) leukocytes;(4) prostate; (5) small intestine; (6) spleen; (7) testis; and (8)thymus.

FIG. 3A. Northern blot analysis of 24P4C12 expression across a panel of,normal human tissues, showing expression of an approximately 3 kbtranscript in kidney. Lanes represent the following tissues: (1) heart;(2) brain; (3) placenta; (4) lung; (5) liver; (6) skeletal muscle; (7)kidney; and (8) pancreas.

FIG. 3B. Northern blot analysis of 24P4C12 expression across a panel ofnormal human tissues, showing expression of an approximately 3 kbtranscript in prostate and colon. Lanes represent the following tissues:(1) spleen; (2) thymus; (3) prostate; (4) testis; (5) ovary; (6) smallintestine; (7) colon; and (8) leukocytes.

FIG. 3C. Northern blot analysis of 24P4C12 expression in prostate cancerxenografts and prostate cancer cell lines. Lanes represent the followingtissues: (1) PrEC; (2) LAPC-4 AD; (3) LAPC-4 AI; (4) LAPC-9 AD; (5)LAPC-9 AI; (6) LNCaP; (7) PC-3; (8) DU145; (9) TsuPr1; and (10) LAPC-4CL.

FIGS. 4A–4B. Amino acid sequence alignment of the 24P4C12 gene productand murine NG22 (SEQ ID NO: 5).

FIG. 5. Expression of 24P4C12 in LAPC xenografts. RNA was extracted fromthe LAPC xenograft that were grown subcutaneously (sc) or intra-tibially(it) within the mouse bone. Northern blots with 10 μg of total RNA/lanewere probed with the 24P4C12 SSH fragment. Size standards in kilobases(kb) are indicated on the side. Lanes represent the following tissues:(1) LAPC-4 AD sc; (2) LAPC-4 AD sc; (3) LAPC-4 AD sc; (4) LAPC-4 AD it;(5) LAPC-4 AD it; (6) LAPC-4 AD it; (7) LAPC-4 AD²; (8) LAPC-9 AD sc;(9) LAPC-9 AD sc; (10) LAPC-9 AD it; (11) LAPC-9 AD it; (12) LAPC-9 ADit; (13) LAPC-3 AI sc; and (14) LAPC-3 AI sc.

FIG. 6A. Expression of 24P4C12 in prostate cancer patient samples. RNAwas extracted from the prostate tumors and their normal adjacent tissuederived from prostate cancer patients. Northern blots with 10 μg oftotal RNA/lane were probed with the 24P4C12 SSH fragment. Size standardsin kilobases (kb) are indicated on the side. Lanes represent thefollowing tissues: (1) Patient 1, normal adjacent tissue; (2) Patient 1,Gleason 7 tumor; (3) Patient 2, normal adjacent tumor; (4) Patient 2,Gleason 9 tumor; (5) Patient 3, normal adjacent tissue; (6) Patient 3,Gleason 7 tumor.

FIG. 6B. Expression of 24P4C12 in prostate cancer patient samples asdescribed for FIG. 6A was compared to β-actin. Lanes represent thefollowing tissues: (1) Patient 1, normal adjacent tissue; (2) Patient 1,Gleason 7 tumor; (3) Patient 2, normal adjacent tumor; (4) Patient 2,Gleason 9 tumor; (5) Patient 3, normal adjacent tissue; (6) Patient 3,Gleason 7 tumor.

FIGS. 7A–7D. The cDNA (SEQ ID NO: 6) and amino acid (SEQ ID NO: 7)sequence of H38087 (clone GTB6). A GC rich (87% GC content) region inthe 5′ untranslated (UTR) region is shown prior to the potential Kozaksequence and start methionine, which are indicated in bold. Thepotential transmembrane domains are underlined in bold.

FIG. 8. Homology alignment of 24P4C12 with H38087 using the BLASTfunction (NCBI).

FIG. 9A. Expression of 24P4C12 in human tissues. A multiple tissuenorthern blot (Clontech) with 2 μg of mRNA/lane was probed with the24P4C12 SSH fragment. Size standards in kilobases (kb) are indicated onthe side. Lanes represent the following tissues: (1) heart; (2) brain;(3) placenta; (4) lung; (5) liver; (6) skeletal muscle; (7) kidney; and(8) pancreas.

FIG. 9B. Expression of 24P4C12 in human tissues. A multiple tissuenorthern blot (Clontech) with 2 μg of mRNA/lane was probed with the24P4C12 SSH fragment. Size standards in kilobases (kb) are indicated onthe side. Lanes represent the following tissues: (1) spleen; (2) thymus;(3) prostate; (4) testis; (5) ovary; (6) small intestine; (7) colon; and(8) leukocytes.

FIG. 9C. Expression of 24P4C12 in human tissues. An LAPC xenograftnorthern blot with 10 μg of total RNA/lane was probed with the 24P4C12SSH fragment. Size standards in kilobases (kb) are indicated on theside. Lanes represent the following tissues: (1) PC-3; (2) LAPC-4 AD;(3) LAPC4 AI; (4) LAPC-9 AD; (5) LAPC-9 AI.

FIG. 10A. Detection of 24P4C12 protein in 293T cells transfected with24P4C12 cDNA by 24P4C12-specific polyclonal antibodies. 293T cells weretransiently transfected with empty vector, or 24P4C12 cDNA in pCDNA 3.1CMV-driven MYC-His or pSR-alpha retroviral expression vectors. Celllysates in sample buffer were then subjected to mild heat denaturation(70° C.) and separated on a 10% SDS-PAGE gel and transferred tonitrocellulose. Membranes were then subjected to western analysis with 2μg/ml of an affinity purified rabbit anti-peptide pAb raised to aminoacids 1–14 (MGGKQRDEDDEAYG) of 24P4C12. Anti-24P4C12 immunoreactivebands were visualized by incubation with anti-rabbit-HRP conjugatedsecondary antibody and enhanced chemiluminescence detection. Resultsshow specific recognition of a 90 kD immunoreactive band (arrow) and ahigh molecular weight smear (>132 Kd) that is enhanced by heatdenaturation.

FIG. 10B. Detection of 24P4C12 protein in 293T cells transfected with24P4C12 cDNA by 24P4C12-specific polyclonal antibodies. Transfected 293Tcells prepared as for FIG. 10A were lysed in sample buffer and subjectedto strong heat denaturation (100° C.). Western analysis with 2 μg/ml ofan affinity purified rabbit anti-peptide pAb raised to amino acids 1–14(MGGKQRDEDDEAYG) of 24P4C12 was performed as for FIG. 10A. Results showspecific recognition of a 90 kD immunoreactive band (arrow) and a highmolecular weight smear (>132 Kd) that is enhanced by heat denaturation.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1–C2 disease under the Whitmore-Jewettsystem, and stage T3–T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C., and most preferably tostringent hybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium. citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37–50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative positions that are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions that are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460–480 (1996): http://blast.wustl/edu/blast/README.html). Furtherdetails regarding amino acid substitutions, which are consideredconservative under such criteria, are provided below.

Additional definitions are provided throughout the subsections thatfollow.

The present invention relates to a novel family of genes and proteins,characterized by multiple transmembrane regions and expression inprostate cancer. More particularly, the invention provides novel genesand proteins, designated 24P4C12 and H38087. The invention is based, inpart, on the identification of the 24P4C12 and H38087 genes and on thecharacterization of the 24P4C12 and H38087 gene expression patterns inprostate cancer, normal prostate, and other normal human tissues. Asdescribed more fully in the examples that follow, the expression patternof the 24P4C12 and H38087 genes was analyzed by: (1) differentialexpression analysis by RT-PCR using target cDNAs prepared from a panelof tissues and cell lines including normal prostate, and the LAPC-4 ADand AI, and LAPC-9 AD xenografts, (2) tissue specificity analysis byRT-PCR using cDNAs prepared from 16 normal human tissues, and (3)northern blot analysis of normal prostate and prostate cancer xenograftsamples. This combined expression analysis was designed to provideinformation on differential expression between AD and AI tissue,clinical prostate cancer and normal prostate, and tissue specificity. Inaddition, initial biological characterization of the 24P4C12 and H38087gene products was undertaken by comparative sequence analysis.

Nucleotide probes corresponding to all or part of the 24P4C12 and H38087cDNAs and gene sequences disclosed herein are provided and may be usedto isolate or identify other cDNAs encoding all or part of the 24P4C12and H38087 gene sequences. The invention further provided primerscapable of specifically amplifying the 24P4C12 and H38087 genes or theirRNA transcripts, and for differentiating between 24P4C12 and H38087molecules. The invention further provides isolated polynucleotidescontaining coding sequences of the 24P4C12 and H38087 gene product(s).Such polynucleotides may be used to express 24P4C12 and H38087 encodedproteins and peptides having a number of further uses. 24P4C12 andH38087 gene probes and primers may also be used to detect the presenceor absence of 24P4C12 and H38087 mRNA in various biological samples, fordetecting prostate cancer cells and other cells expressing 24P4C12 andH38087, and in molecular diagnostic and prognostic assays for prostatecancer. Polynucleotides corresponding or complementary to the 24P4C12gene may be useful in methods for treating prostate cancer, such as, forexample, in modulating or inhibiting 24P4C12 biological activity.

The invention also provides 24P4C12 and H38087 proteins and polypeptidesthat may be used, for example, to generate antibodies. Antibodiescapable of specifically binding to and identifying 24P4C12 and H38087proteins or polypeptides may be used to detect the expression of 24P4C12and H38087, determine their subcellular location, detect and imageprostate cancer cells and prostate tumors, and modulate or inhibit24P4C12 and H38087 biological activity. These and other aspects of theinvention are described in greater detail in the subsections thatfollow.

Structure and Expression of 24P4C12

As is further described in the Examples that follow, the 24P4C12 genesand proteins have been characterized using a number of analyticalapproaches. For example, analyses of nucleotide coding and amino acidsequences were conducted in order to identify potentially relatedmolecules, as well as recognizable structural domains, topologicalfeatures, and other elements within the 24P4C12 mRNA and proteinstructures. Northern blot analyses of 24P4C12 mRNA expression wasconducted in order to establish the range of normal and canceroustissues expressing 24P4C12 message.

The nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2)sequences of an approximately 3 kb 24P4C12 combined cDNA sequence areprovided in FIGS. 1A–1D. This 2587 nucleotide sequence encodes a proteinof 710 amino acids, which contains 13 putative transmembrane domains(underlined in FIGS. 1A–1D, and numbered therein as 105–173, 261–329,439–506, 678–746, 768–836, 924–992, 1074–1142, 1245–1313, 1344–1412,1506–1575, 1694–1763, 1803–1871, and 1935–2000). Comparative sequenceanalysis identified two known genes with significant homology to the24P4C12 cDNA sequence, the recently identified murine NG22 gene and theC. elegans gene designated CEESB82F. Both of these genes encode proteinscontaining 12 transmembrane domains. The murine NG22 gene FIGS. 4A–4B;SEQ ID NO: 5) was recently identified as one of many ORFs within agenomic BAC clone that encompasses the MHC class III in the mousegenome.

Northern blot analysis using an 24P4C12 SSH fragment probe performed on16 normal tissues showed expression primarily in prostate and colon,with lower expression detected in kidney, and significantly lowerexpression detected in pancreas, lung and placenta (FIGS. 2A–2C, 3A–3B).To analyze 24P4C12 expression in cancer tissues northern blotting wasperformed on RNA derived from the LAPC xenografts, and several prostateand non-prostate cancer cell lines. The results show high expressionlevels of 24P4C12 in LAPC-4 AD, LAPC-4 AI, LAPC-9 AD, LNCaP and LAPC-4cell line (FIGS. 2A, 3C, 5). Very high levels are detected in LAPC-3 AI(FIG. 5). Lower levels are detected in LAPC-9 AI (FIG. 3C). Moredetailed analysis of the xenografts shows that 24P4C12 is highlyexpressed in the xenografts even when grown within the tibia of mice(FIG. 5). Northern analysis also shows that 24P4C12 is expressed in thenormal prostate and prostate tumor tissues derived from prostate cancerpatients (FIG. 6A). These results suggest that 24P4C12 is a prostategene that is highly expressed in prostate cancer and may have a utilityas a drug or antibody target in prostate cancer.

Structure and Expression of H38087

H38087 was identified as a family member of 24P4C12 by searching thedBEST database with the 24P4C12 amino acid sequence using the tblastntool in NCBI. ESTs that encode protein fragments of homologous proteinswere identified. One of these, H38087, was cloned from a testis library.The cDNA (clone GTB6) is 2738 bp in size (SEQ ID NO: 6) and encodes a704 amino acid protein (SEQ ID NO: 7) with 11 putative transmembranedomains (underlined in FIGS. 7A–7D, and numbered therein as 152–220,311–379, 743–811, 830–895, 995–1060, 1133–1201, 1394–1459, 1556–1624,1655–1723, 1859–1924, and 19880–2056). The 58 base pairs of 5′untranslated region are very GC rich (87%), indicating that this genemay contain translational regulatory elements. The amino acid sequencesof 24P4C12 and H38087 are 44% identical and 56% homologous over theentire sequence (FIG. 8).

Expression analysis shows that H38087 is ubiquitously expressed (FIG.9), with highest expression levels detected in testis. Expression isalso seen in all the LAPC xenografts. Because H38087 is ubiquitouslyexpressed, it could serve as a control for testing 24P4C12-specifictherapeutics. A 24P4C12-specific therapeutic that affects H38087function could be toxic to normal cells. However, a therapeutic thatselectively affects 24P4C12, but not H38087, may be less toxic to normalcells. Therefore, H38087 protein is useful as a pre-clinical testingtool for therapeutic modalities directed towards 24P4C12.

Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 24P4C12 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 24P4C12 protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a 24P4C12 gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides that hybridize to a 24P4C12 gene,mRNA, or to a 24P4C12 encoding polynucleotide (collectively, “24P4C12polynucleotides”). As used herein, the 24P4C12 gene and protein is meantto include the 24P4C12 genes and proteins specifically described hereinand the genes and proteins corresponding to other 24P4C12 proteins andstructurally similar variants of the foregoing. Such other 24P4C12proteins and variants will generally have coding sequences that arehighly homologous to the 24P4C12 coding sequence, and preferably willshare at least about 50% amino acid identity and at least about 60%amino acid homology (using BLAST criteria), more preferably sharing 70%or greater homology (using BLAST criteria).

One embodiment of a 24P4C12 polynucleotide is a 24P4C12 polynucleotidehaving the sequence shown in FIGS. 1A–1D (SEQ ID NO: 1). A 24P4C12polynucleotide may comprise a polynucleotide having the nucleotidesequence of human 24P4C12 as shown in FIGS. 1A–1D (SEQ ID NO: 1),wherein T can also be U; a polynucleotide that encodes all or part ofthe 24P4C12 protein; a sequence complementary to the foregoing; or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIGS. 1A–1D(SEQ ID NO: 1), from nucleotide residue number 6 through nucleotideresidue number 2138, or having the sequence as shown in FIG. 1E (SEQ IDNO: 3), wherein T can also be U. Another embodiment comprises apolynucleotide encoding a 24P4C12 polypeptide whose sequence is encodedby the cDNA contained in either of the plasmids designated p24P4C12-GTE5or p24P4C12-GTE9 deposited with American Type Culture Collection asDesignation Nos. 207129 and 207084, respectively. Another embodimentcomprises a polynucleotide that is capable of hybridizing understringent hybridization conditions to the human 24P4C12 cDNA shown inFIGS. 1A–1D (SEQ ID NO: 1) or to a polynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include 24P4C12polynucleotides encoding specific portions of the 24P4C12 mRNA sequencesuch as those that encode the protein and fragments thereof. Forexample, representative embodiments of the invention disclosed hereininclude: polynucleotides encoding about amino acid 1 to about amino acid10 of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2),polynucleotides encoding about amino acid 20 to about amino acid 30 ofthe 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2), polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 24P4C12protein shown in FIGS. 1A–1D (SEQ ID NO: 2), polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 24P4C12 protein shownin FIGS. 1A–1D (SEQ ID NO: 2), polynucleotides encoding about amino acid50 to about amino acid 60 of the 24P4C12 protein shown in FIGS. 1A–1D(SEQ ID NO: 2), polynucleotides encoding about amino acid 60 to aboutamino acid 70 of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO:2), polynucleotides encoding about amino acid 70 to about amino acid 80of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2),polynucleotides encoding about amino acid 80 to about amino acid 90 ofthe 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2) andpolynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2), etc. Followingthis scheme, polynucleotides (of at least 10 amino acids) encodingportions of the amino acid sequence of amino acids 100–710 of the24P4C12 protein are typical embodiments of the invention.Polynucleotides encoding larger portions of the 24P4C12 protein are alsocontemplated. For example polynucleotides encoding from about amino acid1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50etc.) of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2) may begenerated by a variety of techniques well known in the art.

Additional illustrative embodiments of the invention disclosed hereininclude 24P4C12 polynucleotide fragments encoding one or more of thebiological motifs contained within the 24P4C12 protein sequence. In oneembodiment, typical polynucleotide fragments of the invention can encodeone or more of the transmembrane domains disclosed herein. In anotherembodiment, typical polynucleotide fragments of the invention can encodeone or more of the regions of 24P4C12 that exhibit homology to H38087,NG22 or CEESB82F. In another embodiment of the invention, typicalpolynucleotide fragments can encode one or more of the 24P4C12N-glycosylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase phosphorylation, N-myristoylation, oramidation sites, or the leucine zipper pattern, as disclosed in greaterdetail in the text discussing the 24P4C12 protein and polypeptidesbelow. In yet another embodiment of the invention, typicalpolynucleotide fragments can encode sequences that are unique to one ormore 24P4C12 alternative splicing variants.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. For example, as 24P4C12 is shown to bespecifically expressed in prostate cancers (FIGS. 2A, 3C, 5, 6), thesepolynucleotides may be used in methods for assessing the status of24P4C12 gene products in normal versus cancerous tissues. Typically,polynucleotides encoding specific regions of the 24P4C12 protein may beused to assess the presence of perturbations (such as deletions,insertions, point mutations etc.) in specific regions (such regionscontaining a transmembrane domain) of the 24P4C12 gene products.Exemplary assays include both RT-PCR assays as well as single-strandconformation polymorphism (SSCP) analysis (see e.g. Marrogi et al., J.Cutan. Pathol. 26(8): 369–378 (1999), both of which utilizepolynucleotides encoding specific regions of a protein to examine theseregions within the protein.

Likewise, the invention additionally provides polynucleotidescorresponding or complementary to all or part of a H38087 gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding a H38087 protein and fragments thereof, DNA,RNA, DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a H38087 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa H38087 gene, mRNA, or to a H38087 encoding polynucleotide(collectively, “H38087 polynucleotides”). As used herein, the H38087gene and protein is meant to include the H38087 genes and proteinsspecifically described herein and the genes and proteins correspondingto other H38087 proteins and structurally similar variants of theforegoing. Such other H38087 proteins and variants will generally havecoding sequences that are highly homologous to the H38087 codingsequence, and preferably will share at least about 50% amino acididentity and at least about 60% amino acid homology (using BLASTcriteria), more preferably sharing 70% or greater homology (using BLASTcriteria).

One embodiment of a H38087 polynucleotide is a H38087 polynucleotidehaving the sequence shown in FIGS. 7A–7D (SEQ ID NO: 6). A H38087polynucleotide may comprise a polynucleotide having the nucleotidesequence of human H38087 as shown in FIGS. 7A–7D (SEQ ID NO: 6), whereinT can also be U; a polynucleotide that encodes all or part of the H38087protein; a sequence complementary to the foregoing; or a polynucleotidefragment of any of the foregoing. Another embodiment comprises apolynucleotide having the sequence as shown in FIGS. 7A–7D (SEQ ID NO:6), from nucleotide residue number 59 through nucleotide residue number2173 (using the numbering shown in FIGS. 7A–7D; SEQ ID NO: 6). Anotherembodiment comprises a polynucleotide that is capable of hybridizingunder stringent hybridization conditions to the human H38087 cDNA shownin FIGS. 7A–7D or to a polynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include H38087polynucleotides encoding specific portions of the H38087 mRNA sequencesuch as those that encode the protein and fragments thereof. Forexample, representative embodiments of the invention disclosed hereininclude: polynucleotides encoding about amino acid 1 to about amino acid10 of the H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7),polynucleotides encoding about amino acid 20 to about amino acid 30 ofthe H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polynucleotidesencoding about amino acid 30 to about amino acid 40 of the H38087protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polynucleotides encodingabout amino acid 40 to about amino acid 50 of the H38087 protein shownin FIGS. 7A–7D (SEQ ID NO: 7), polynucleotides encoding about amino acid50 to about amino acid 60 of the H38087 protein shown in FIGS. 7A–7D(SEQ ID NO: 7), polynucleotides encoding about amino acid 60 to aboutamino acid 70 of the H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7),polynucleotides encoding about amino acid 70 to about amino acid 80 ofthe H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polynucleotidesencoding about amino acid 80 to about amino acid 90 of the H38087protein shown in FIGS. 7A–7D (SEQ ID NO: 7) and polynucleotides encodingabout amino acid 90 to about amino acid 100 of the H38087 protein shownin FIGS. 7A–7D (SEQ ID NO: 7), etc. Following this scheme,polynucleotides (of at least 10 amino acids) encoding portions of theamino acid sequence of amino acids 100–704 of the H38087 protein aretypical embodiments of the invention. Polynucleotides encoding largerportions of the H38087 protein are also contemplated. For examplepolynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the H38087protein shown in FIGS. 7A–7D (SEQ ID NO: 7) may be generated by avariety of techniques well known in the art.

Additional illustrative embodiments of the invention disclosed hereininclude H38087 polynucleotide fragments encoding one or more of thebiological motifs contained within the H38087 protein sequence. In oneembodiment, typical polynucleotide fragments of the invention can encodeone or more of the transmembrane domains disclosed herein. In anotherembodiment, typical polynucleotide fragments of the invention can encodeone or more of the regions of H38087 that exhibit homology to 24P4C12,NG22 or CEESB82F. In another embodiment of the invention, typicalpolynucleotide fragments can encode one or more of the H38087N-glycosylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase phosphorylation, or N-myristoylationsites, or the signal sequence, as disclosed in greater detail in thetext discussing the H38087 protein and polypeptides below. In yetanother embodiment of the invention, typical polynucleotide fragmentscan encode sequences that are unique to one or more H38087 alternativesplicing variants.

Other specifically contemplated embodiments of the invention disclosedherein are genomic DNA, cDNAs, ribozymes, and antisense molecules, aswell as nucleic acid molecules based on an alternative backbone orincluding alternative bases, whether derived from natural sources orsynthesized. For example, antisense molecules can be RNAs or othermolecules, including peptide nucleic acids (PNAs) or non-nucleic acidmolecules such as phosphorothioate derivatives, that specifically bindDNA or RNA in a base pair-dependent manner. A skilled artisan canreadily obtain these classes of nucleic acid molecules using the 24P4C12and H38087 polynucleotides and polynucleotide sequences disclosedherein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,24P4C12 or H38087. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES,Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis1:1–5 (1988). The 24P4C12 and H38087 antisense oligonucleotides of thepresent invention include derivatives such as S-oligonucleotides(phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra),which exhibit enhanced cancer cell growth inhibitory action. S-oligos(nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfurtransfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693–4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253–1254 (1990),the disclosures of which are fully incorporated by reference herein.

The 24P4C12 and H38087 antisense oligonucleotides of the presentinvention typically may be RNA or DNA that is complementary to andstably hybridizes with the first 100 N-terminal codons or last 100C-terminal codons of the 24P4C12 or H38087 genome or the correspondingmRNA. While absolute complementarity is not required, high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 24P4C12 orH38087 mRNA and not to mRNA specifying other regulatory subunits ofprotein kinase. Preferably, the 24P4C12 and H38087 antisenseoligonucleotides of the present invention are a 15 to 30-mer fragment ofthe antisense DNA molecule having a sequence that hybridizes to 24P4C12or H38087 mRNA. Optionally, the 24P4C12 or H38087 antisenseoligonucleotide is a 30-mer oligonucleotide that is complementary to aregion in the first 10 N-terminal codons and last 10 C-terminal codonsof 24P4C12 or H38087. Alternatively, the antisense molecules aremodified to employ ribozymes in the inhibition of 24P4C12 or H38087expression. L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510–515(1996).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a 24P4C12 or H38087 polynucleotide in a sampleand as a means for detecting a cell expressing a 24P4C12 or H38087protein.

Examples of such probes include polypeptides comprising all or part ofthe human 24P4C12 cDNA sequence shown in FIGS. 1A–1D (SEQ ID NO: 1) orthe human H38087 cDNA sequence FIGS. 7A–7D (SEQ ID NO: 6). Examples ofprimer pairs capable of specifically amplifying 24P4C12 or H38087 mRNAsare also described in the Examples that follow. As will be understood bythe skilled artisan, a great many different primers and probes may beprepared based on the sequences provided herein and used effectively toamplify and/or detect a 24P4C12 or H38087 mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the 24P4C12 or H38087 gene orthat encode polypeptides other than 24P4C12 or H38087 gene product orfragments thereof. A skilled artisan can readily employ nucleic acidisolation procedures to obtain an isolated 24P4C12 or H38087polynucleotide.

The 24P4C12 or H38087 polynucleotides of the invention are useful for avariety of purposes, including but not limited to their use as probesand primers for the amplification and/or detection of the 24P4C12 orH38087 genes, mRNAs, or fragments thereof; as reagents for the diagnosisand/or prognosis of prostate cancer and other cancers; as codingsequences capable of directing the expression of 24P4C12 or H38087polypeptides; as tools for modulating or inhibiting the expression ofthe 24P4C12 or H38087 genes and/or translation of the 24P4C12 or H38087transcripts; and as therapeutic agents.

Isolation of 24P4C12-and H38087-Encoding Nucleic Acid Molecules

The 24P4C12 and H38087 cDNA sequences described herein enable theisolation of other polynucleotides encoding 24P4C12 or H38087 geneproduct(s), as well as the isolation of polynucleotides encoding 24P4C12or H38087 gene product homologs, alternatively spliced isoforms, allelicvariants, and mutant forms of the 24P4C12 or H38087 gene product.Various molecular cloning methods that can be employed to isolate fulllength cDNAs encoding a 24P4C12 or H38087 gene are well known (See, forexample, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2dedition., Cold Spring Harbor Press, New York, 1989; Current Protocols inMolecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). Forexample, lambda phage cloning methodologies may be convenientlyemployed, using commercially available cloning systems (e.g., Lambda ZAPExpress, Stratagene). Phage clones containing 24P4C12 or H38087 genecDNAs may be identified by probing with a labeled 24P4C12 or H38087 cDNAor a fragment thereof. For example, in one embodiment, the 24P4C12 cDNA(FIGS. 1A–1D; SEQ ID NO: 1) or a portion thereof can be synthesized andused as a probe to retrieve overlapping and full length cDNAscorresponding to a 24P4C12 gene. The 24P4C12 gene itself may be isolatedby screening genomic DNA libraries, bacterial artificial chromosomelibraries (BACs), yeast artificial chromosome libraries (YACs), and thelike, with 24P4C12 DNA probes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 24P4C12 or H38087 polynucleotide, including but not limited to phages,plasmids, phagemids, cosmids, YACs, BACs, as well as various viral andnon-viral vectors well known in the art, and cells transformed ortransfected with such recombinant DNA or RNA molecules. As used herein,a recombinant DNA or RNA molecule is a DNA or RNA molecule that has beensubjected to molecular manipulation in vitro. Methods for generatingsuch molecules are well known (see, for example, Sambrook et al, 1989,supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 24P4C12 and/or H38087polynucleotide within a suitable prokaryotic or eukaryotic host cell.Examples of suitable eukaryotic host cells include a yeast cell, a plantcell, or an animal cell, such as a mammalian cell or an insect cell(e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell).Examples of suitable mammalian cells include various prostate cancercell lines such LnCaP, PC-3, DU145, LAPC-4, TsuPr1, other transfectableor transducible prostate cancer cell lines, as well as a number ofmammalian cells routinely used for the expression of recombinantproteins (e.g., COS, CHO, 293, 293T cells). More particularly, apolynucleotide comprising the coding sequence of 24P4C12 or H38087 maybe used to generate 24P4C12 or H38087 proteins or fragments thereofusing any number of host-vector systems routinely used and widely knownin the art.

A wide range of host-vector systems suitable for the expression of24P4C12 and H38087 proteins or fragments thereof are available, see forexample, Sambrook et al., 1989, supra; Current Protocols in MolecularBiology, 1995, supra). Preferred vectors for mammalian expressioninclude but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) andthe retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785).Using these expression vectors, 24P4C12 or H38087 may be preferablyexpressed in several prostate cancer and non-prostate cell lines,including for example 293, 293T, rat-1, NIH 3T3, PC-3, LNCaP and TsuPr1.The host-vector systems of the invention are useful for the productionof a 24P4C12 or H38087 protein or fragment thereof. Such host-vectorsystems may be employed to study the functional properties of 24P4C12 orH38087 and 24P4C12 or H38087 mutations.

Recombinant human 24P4C12 or H38087 protein may be produced by mammaliancells transfected with a construct encoding 24P4C12 or H38087. In anillustrative embodiment described in the Examples, 293T cells can betransfected with an expression plasmid encoding 24P4C12, the 24P4C12protein is expressed in the 293T cells, and the recombinant 24P4C12protein can be isolated using standard purification methods (e.g.,affinity purification using anti-24P4C12 antibodies). In anotherembodiment, also described in the Examples herein, the 24P4C12 codingsequence is subcloned into the retroviral vector pSRαtkneo and used toinfect various mammalian cell lines, such as NIH 3T3, PC3 and LnCaP inorder to establish 24P4C12 expressing cell lines. Various otherexpression systems well known in the art may also be employed.Expression constructs encoding a leader peptide joined in frame to the24P4C12 coding sequence may be used for the generation of a secretedform of recombinant 24P4C12 protein.

Proteins encoded by the 24P4C12 or H38087 genes, or by fragmentsthereof, will have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 24P4C12 or H38087 geneproduct. Antibodies raised against a 24P4C12 or H38087 protein orfragment thereof may be useful in diagnostic and prognostic assays, andimaging methodologies in the management of human cancers characterizedby expression of 24P4C12 protein, including but not limited to cancer ofthe prostate. Such antibodies may be expressed intracellularly and usedin methods of treating patients with such cancers. Various immunologicalassays useful for the detection of 24P4C12 and H38087 proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting 24P4C12 or H38087expressing cells (e.g., in radioscintigraphic imaging methods). 24P4C12proteins may also be particularly useful in generating cancer vaccines,as further described below.

24P4C12 Polypeptides

Another aspect of the present invention provides 24P4C12 proteins andpolypeptide fragments thereof. The 24P4C12 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined below. Fusion proteins that combine partsof different 24P4C12 proteins or fragments thereof, as well as fusionproteins of a 24P4C12 protein and a heterologous polypeptide are alsoincluded. Such 24P4C12 proteins will be collectively referred to as the24P4C12 proteins, the proteins of the invention, or 24P4C12. As usedherein, the term “24P4C12 polypeptide” refers to a polypeptide fragmentor a 24P4C12 protein of at least 10 amino acids, preferably at least 15amino acids.

Specific embodiments of 24P4C12 proteins comprise a polypeptide havingthe amino acid sequence of human 24P4C12 as shown in FIGS. 1A–1D (SEQ IDNO: 2). Alternatively, embodiments of 24P4C12 proteins comprise variantpolypeptides having alterations in the amino acid sequence of human24P4C12 as shown in FIGS. 1A–1D (SEQ ID NO: 2).

In general, naturally occurring allelic variants of human 24P4C12 willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the 24P4C12 proteins willcontain conservative amino acid substitutions within the 24P4C12sequences described herein or will contain a substitution of an aminoacid from a corresponding position in a 24P4C12 homologue. One class of24P4C12 allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular 24P4C12 amino acidsequence, but will further contain a radical departure form thesequence, such as a non-conservative substitution, truncation, insertionor frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of 24P4C12 proteins such as polypeptides havingamino acid insertions, deletions and substitutions. 24P4C12 variants canbe made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller etal., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells etal., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other knowntechniques can be performed on the cloned DNA to produce the 24P4C12variant DNA. Scanning amino acid analysis can also be employed toidentify one or more amino acids along a contiguous sequence. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions[Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol.Biol., 150:1 (1976)]. If alanine substitution does not yield adequateamounts of variant, an isosteric amino acid can be used.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 710 amino acid sequence of the24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of the 24P4C12protein shown in FIGS. 1A–1D (SEQ ID NO: 2), polypeptides consisting ofabout amino acid 30 to about amino acid 40 of the 24P4C12 protein shownin FIGS. 1A–1D (SEQ ID NO: 2), polypeptides consisting of about aminoacid 40 to about amino acid 50 of the 24P4C12 protein shown in FIGS.1A–1D (SEQ ID NO: 2), polypeptides consisting of about amino acid 50 toabout amino acid 60 of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ IDNO: 2), polypeptides consisting of about amino acid 60 to about aminoacid 70 of the 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2),polypeptides consisting of about amino acid 70 to about amino acid 80 ofthe 24P4C12 protein shown in FIGS. 1A–1D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the 24P4C12protein shown in FIGS. 1A–1D (SEQ ID NO: 2) and polypeptides consistingof about amino acid 90 to about amino acid 100 of the 24P4C12 proteinshown in FIGS. 1A–1D (SEQ ID NO: 2), etc. Following this scheme,polypeptides consisting of portions of the amino acid sequence of aminoacids 100–710 of the 24P4C12 protein are typical embodiments of theinvention. Polypeptides consisting of larger portions of the 24P4C12protein are also contemplated. For example polypeptides consisting ofabout amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or30, or 40 or 50 etc.) of the 24P4C12 protein shown in FIGS. 1A–1D (SEQID NO: 2) may be generated by a variety of techniques well known in theart.

Additional illustrative embodiments of the invention disclosed hereininclude 24P4C12 polypeptides containing the amino acid residues of oneor more of the biological motifs contained within the 24P4C12polypeptide sequence as shown in FIG. 1A (SEQ ID NO: 2). In oneembodiment, typical polypeptides of the invention can contain one ormore of the transmembrane regions shown in FIGS. 1A–1D (SEQ ID NO: 2),or one or more of the regions of 24P4C12 that exhibit homology toH38087, NG22 OR CEESB82F. In another embodiment, typical polypeptides ofthe invention can contain one or more of the 24P4C12 N-glycosylationsites such as NRSC (SEQ ID NO: 8) at residues 29–32 (numbering fromfirst amino acid residue shown in FIG. 1A), NSTG (SEQ ID NO: 9) atresidues 69–72, NMTV (SEQ ID NO: 10) at residues 155–158, NDTT (SEQ IDNO: 11) at residues 197–200, NLSA (SEQ ID NO: 12) at residues 298–301,NISS (SEQ ID NO: 13) at residues 393–396, NTSC (SEQ ID NO: 14) atresidues 405–408, NSSC (SEQ ID NO: 15) at residues 416–419, and/or NGSL(SEQ ID NO: 16) at residues 678–681. In another embodiment, typicalpolypeptides of the invention can contain one or more of the 24P4C12protein kinase C phosphorylation sites such as SFR at residues 22–24,SVK at residues 218–220, SSK at residues 430–432, TLR at residues494–496, SAK at residues 573–575, and/or SGR at residues 619–621. Inanother embodiment, typical polypeptides of the invention can containone or more of the 24P4C12 casein kinase II phosphorylation sites suchas SCTD (SEQ ID NO: 17) at residues 31–34, SVAE (SEQ ID NO: 18) atresidues 102–105, SCPE (SEQ ID NO: 19) at residues 119–122, TVGE (SEQ IDNO: 20) at residues 135–138, and/or SVQE (SEQ ID NO: 21) at residues304–307. In another embodiment, typical polypeptides of the inventioncan contain one or more of the tyrosine kinase phosphorylation sitessuch as RDEDDEAY (SEQ ID NO: 22) at residues 6–13. In anotherembodiment, typical polypeptides of the invention can contain one ormore of the N-myristoylation sites such as GAYCGM (SEQ ID NO: 23) atresidues 72–77, GMGENK (SEQ ID NO: 24) at residues 76–81, GVPWNM (SEQ IDNO: 25) at residues 151–156, GLIDSL (SEQ ID NO: 26) at residues 207–212,GIYYCW (SEQ ID NO: 27) at residues 272–277, GASISQ (SEQ ID NO: 28) atresidues 287–292, GQMMST (SEQ ID NO: 29) at residues 379–354, GLFWTL(SEQ ID NO: 30) at residues 449–454, and/or GAFASF (SEQ ID NO: 31) atresidues 467–472. In another embodiment, typical polypeptides of theinvention can contain one or more of the amidation sites such as LGKK(SEQ ID NO: 32) at residues 695–698. In another embodiment, typicalpolypeptides of the invention can contain a leucine zipper pattern suchas LFILLLRLVAGPLVLVILGVL (SEQ ID NO: 33) at residues 245–266. Relatedembodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those which contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides.

In yet another embodiment of the invention, typical polypeptides cancontain amino acid sequences that are unique to one or more 24P4C12alternative splicing variants. The monitoring of alternative splicevariants of 24P4C12 is useful because changes in the alternativesplicing of proteins is suggested as one of the steps in a series ofevents that lead to the progression of cancers (see e.g. Carstens etal., Oncogene 15(250: 3059–3065 (1997)). Consequently, monitoring ofalternative splice variants of 24P4C12 provides an additional means toevaluate syndromes associated with perturbations in 24P4C12 geneproducts such as cancers.

Polypeptides consisting of one or more of the 24P4C12 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 24P4C12 motifsdiscussed above are associated with growth disregulation and because24P4C12 is overexpressed in cancers (FIG. 5). Casein kinase II and cAMPand cCMP-dependent protein kinase, for example are enzymes known to beassociated with the development of the malignant phenotype (see e.g.Chen et al., Lab Invest., 78(2): 165–174 (1998); Gaiddon et al.,Endocrinology 136(10): 4331–4338 (1995) and Hall et al., Nucleic AcidsResearch 24(6): 1119–1126 (1996)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochim. Biophys. Acta1473(1):21–34 (1999); Raju et al., Exp. Cell Res. 235(1): 145–154(1997)).

The polypeptides of the preceding paragraphs have a number of differentspecific uses. As 24P4C12 is shown to be highly expressed in prostatecancers (FIGS. 2A, 3C, 5, 6), these polypeptides may be used in methodsfor assessing the status of 24P4C12 gene products in normal versuscancerous tissues and elucidating the malignant phenotype. Typically,polypeptides encoding specific regions of the 24P4C12 protein may beused to assess the presence of perturbations (such as deletions,insertions, point mutations etc.) in specific regions (such as regionscontaining a transmembrane domain) of the 24P4C12 gene products.Exemplary assays can utilize antibodies targeting a 24P4C12 polypeptidescontaining the amino acid residues of one or more of the biologicalmotifs contained within the 24P4C12 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues. Alternatively, 24P4C12 polypeptides containing the amino acidresidues of one or more of the biological motifs contained within the24P4C12 polypeptide sequence can be used to screen for factors thatinteract with that region of 24P4C12.

As discussed above, redundancy in the genetic code permits variation in24P4C12 gene sequences. In particular, one skilled in the art willrecognize specific codon preferences by a specific host species and canadapt the disclosed sequence as preferred for a desired host. Forexample, preferred codon sequences typically have rare codons (i.e.,codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific organism may be calculated, forexample, by utilizing codon usage tables available on the INTERNET atthe following address: http://www.dna.affrc.go.jp/˜nakamura/codon.html.Nucleotide sequences that have been optimized for a particular hostspecies by replacing any codons having a usage frequency of less thanabout 20% are referred to herein as “codon optimized sequences.”

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that may be deleterious to gene expression. The GC content ofthe sequence may be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostcell. Where possible, the sequence may also be modified to avoidpredicted hairpin secondary mRNA structures. Other useful modificationsinclude the addition of a translational initiation consensus sequence atthe start of the open reading frame, as described in Kozak, Mol. CellBiol., 9:5073–5080 (1989). Nucleotide sequences that have been optimizedfor expression in a given host species by elimination of spuriouspolyadenylation sequences, elimination of exon/intron splicing signals,elimination of transposon-like repeats and/or optimization of GC contentin addition to codon optimization are referred to herein as an“expression enhanced sequence.”

24P4C12 proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the 24P4C12protein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated 24P4C12 protein. A purified 24P4C12protein molecule will be substantially free of other proteins ormolecules that impair the binding of 24P4C12 to antibody or otherligand. The nature and degree of isolation and purification will dependon the intended use. Embodiments of a 24P4C12 protein include a purified24P4C12 protein and a functional, soluble 24P4C12 protein. In one form,such functional, soluble 24P4C12 proteins or fragments thereof retainthe ability to bind antibody or other ligand.

The invention also provides 24P4C12 polypeptides comprising biologicallyactive fragments of the 24P4C12 amino acid sequence, such as apolypeptide corresponding to part of the amino acid sequence for 24P4C12as shown in FIGS. 1A–1D (SEQ ID NO: 2). Such polypeptides of theinvention exhibit properties of the 24P4C12 protein, such as the abilityto elicit the generation of antibodies that specifically bind an epitopeassociated with the 24P4C12 protein. 24P4C12 polypeptides can begenerated using standard peptide synthesis technology or using chemicalcleavage methods well known in the art based on the amino acid sequencesof the human 24P4C12 proteins disclosed herein. Alternatively,recombinant methods can be used to generate nucleic acid molecules thatencode a polypeptide fragment of a 24P4C12 protein. In this regard, the24P4C12-encoding nucleic acid molecules described herein provide meansfor generating defined fragments of 24P4C12 proteins. 24P4C12polypeptides are particularly useful in generating and characterizingdomain specific antibodies (e.g., antibodies recognizing anextracellular or intracellular epitope of a 24P4C12 protein), inidentifying agents or cellular factors that bind to 24P4C12 or aparticular structural domain thereof, and in various therapeuticcontexts, including but not limited to cancer vaccines.

24P4C12 polypeptides containing particularly interesting structures canbe predicted and/or identified using various analytical techniques wellknown in the art, including, for example, the methods of Chou-Fasman,Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz orJameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-24P4C12 antibodies or in identifying cellular factors thatbind to 24P4C12.

In an embodiment described in the examples that follow, 24P4C12 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 24P4C12 with a C-terminal 6× His and MYC tag(pcDNA3.1/mycHIS, Invitrogen). The HIS-tagged 24P4C12 expressed in cellsmay be purified using a nickel column using standard techniques.

Modifications of 24P4C12 such as covalent modifications are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a 24P4C12 polypeptidewith an organic derivatizing agent that is capable of reacting withselected side chains or the N- or C-terminal residues of the 24P4C12.Another type of covalent modification of the 24P4C12 polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence 24P4C12(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequence24P4C12. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present. Anothertype of covalent modification of 24P4C12 comprises linking the 24P4C12polypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The 24P4C12 of the present invention may also be modified in a way toform a chimeric molecule comprising 24P4C12 fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the 24P4C12 with apolyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the 24P4C12. In analternative embodiment, the chimeric molecule may comprise a fusion ofthe 24P4C12 with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 24P4C12 polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgGI molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

H38087 Polypeptides

Another aspect of the present invention provides H38087 proteins andpolypeptide fragments thereof. The H38087 proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein. Fusion proteins that combineparts of different H38087 proteins or fragments thereof, as well asfusion proteins of a H38087 protein and a heterologous polypeptide arealso included. Such H38087 proteins will be collectively referred to asthe H38087 proteins or H38087. As used herein, the term “H38087polypeptide” refers to a polypeptide fragment or a H38087 protein of atleast 10 amino acids, preferably at least 15 amino acids.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 704 amino acid sequence of theH38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of the H38087protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polypeptides consisting ofabout amino acid 30 to about amino acid 40 of the H38087 protein shownin FIGS. 7A–7D (SEQ ID NO: 7), polypeptides consisting of about aminoacid 40 to about amino acid 50 of the H38087 protein shown in FIGS.7A–7D (SEQ ID NO: 7), polypeptides consisting of about amino acid 50 toabout amino acid 60 of the H38087 protein shown in FIGS. 7A–7D (SEQ IDNO: 7), polypeptides consisting of about amino acid 60 to about aminoacid 70 of the H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7),polypeptides consisting of about amino acid 70 to about amino acid 80 ofthe H38087 protein shown in FIGS. 7A–7D (SEQ ID NO: 7), polypeptidesconsisting of about amino acid 80 to about amino acid 90 of the H38087protein shown in FIGS. 7A–7D (SEQ ID NO: 7) and polypeptides consistingof about amino acid 90 to about amino acid 100 of the H38087 proteinshown in FIGS. 7A–7D (SEQ ID NO: 7), etc. Following this scheme,polypeptides consisting of portions of the amino acid sequence of aminoacids 100–710 of the H38087 protein are typical embodiments of theinvention. Polypeptides consisting of larger portions of the H38087protein are also contemplated. For example polypeptides consisting ofabout amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or30, or 40 or 50 etc.) of the H38087 protein shown in FIGS. 7A–7D (SEQ IDNO: 7) may be generated by a variety of techniques well known in theart.

Additional illustrative embodiments of the invention disclosed hereininclude H38087 polypeptides containing the amino acid residues of one ormore of the biological motifs contained within the H38087 polypeptidesequence as shown in FIG. 7 (SEQ ID NO: 7). In one embodiment, typicalpolypeptides of the invention can contain one or more of thetransmembrane regions shown in FIGS. 7A–7D, or one or more of theregions of H38087 that exhibit homology to 24P4C12, NG22 OR CEESB82F. Inanother embodiment, typical polypeptides of the invention can containone or more of the H38087 N-glycosylation sites such as NETT (SEQ ID NO:46) at residues 185–188 (numbering from first amino acid residue shownin FIG. 7), NITD (SEQ ID NO: 47) at residues 198–201, and/or NKTN (SEQID NO: 48) at residues 695–698. In another embodiment, typicalpolypeptides of the invention can contain one or more of the H38087protein kinase C phosphorylation sites such as TFK at residues 19–21,SSR at residues 126–128, SRK at residues 195–197, TAK at residues402–404, SAR at residues 574–576, THR at residues 620–622, TLK atresidues 689–691, and/or TNK at residues 697–699. In another embodiment,typical polypeptides of the invention can contain one or more of theH38087 casein kinase II phosphorylation sites such as THGD (SEQ ID NO:49) at residues 54–57, SRGE (SEQ ID NO: 50) at residues 67–70, TKNE (SEQID NO: 51) at residues 77–80, SSRD (SEQ ID NO: 52) at residues 126–129,TTYE (SEQ ID NO: 53) at residues 187–190, TYED (SEQ ID NO: 54) atresidues 188–191, SLVD (SEQ ID NO: 55) at residues 293–296, SILE (SEQ IDNO: 56) at residues 321–234, TSNE (SEQ ID NO: 57) at residues 385–388and/or SSHE (SEQ ID NO: 58) at residues 413–416. In another embodiment,typical polypeptides of the invention can contain one or more of thetyrosine kinase phosphorylation sites such as RSSRDFEYY (SEQ ID NO: 59)at residues 125–133. In another embodiment, typical polypeptides of theinvention can contain one or more of the N-myristoylation sites such asGQKGTK (SEQ ID NO: 60) at residues 73–78, GNETTY (SEQ ID NO: 61) atresidues 184–189, GSRKNI (SEQ ID NO: 62) at residues 194–199, GAKKAN(SEQ ID NO: 63) at residues 205–210, GVLEAR (SEQ ID NO: 64) at residues211–216, GLVIAM (SEQ ID NO: 65) at residues 236–241, GIFHCY (SEQ ID NO:66) at residues 273–278, GSDVSL (SEQ ID NO: 67) at residues 289–294,GGESGY (SEQ ID NO: 68) at residues 431–436, GAFASY (SEQ ID NO: 69) atresidues 468–473, and/or GTNFCT (SEQ ID NO: 70) at residues 568–573.Related embodiments of these inventions include polypeptides containingcombinations of the different motifs discussed above with preferableembodiments being those which contain no insertions, deletions orsubstitutions either within the motifs or the intervening sequences ofthese polypeptides.

The H38087 polypeptides of the invention can be modified, generated andused in manners analogous to those described above for 24P4C12polypeptides, as would be known and appreciated by those skilled in theart.

24P4C12 Antibodies

The term “antibody” is used in the broadest sense and specificallycovers single anti-24P4C12 monoclonal antibodies (including agonist,antagonist and neutralizing antibodies) and anti-24P4C12 antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” (mAb) as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiescomprising the individual population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

Another aspect of the invention provides antibodies that bind to 24P4C12proteins and polypeptides. The most preferred antibodies willspecifically bind to a 24P4C12 protein and will not bind (or will bindweakly) to non-24P4C12 proteins and polypeptides. Anti-24P4C12antibodies that are particularly contemplated include monoclonal andpolyclonal antibodies as well as fragments containing the antigenbinding domain and/or one or more complementarity determining regions ofthese antibodies. As used herein, an antibody fragment is defined as atleast a portion of the variable region of the immunoglobulin moleculethat binds to its target, i.e., the antigen binding region.

24P4C12 antibodies of the invention may be particularly useful inprostate cancer diagnostic and prognostic assays, and imagingmethodologies. Intracellularly expressed antibodies (e.g., single chainantibodies) may be therapeutically useful in treating cancers in whichthe expression of 24P4C12 is involved, such as for example advanced andmetastatic prostate cancers. Also useful in therapeutic methods fortreatment of prostate cancer are systemically administered 24P4C12antibodies that interfere with 24P4C12 function or that targetextracellular regions of 24P4C12 for delivery of a toxin or therapeuticmolecule. Such delivery of a toxin or therapeutic molecule can beachieved using known methods of conjugating a second molecule to the24P4C12 antibody or fragment thereof. Similarly, such antibodies may beuseful in the treatment, diagnosis, and/or prognosis of other cancers,to the extent 24P4C12 is also expressed or overexpressed in other typesof cancer.

The invention also provides various immunological assays useful for thedetection and quantification of 24P4C12 and mutant 24P4C12 proteins andpolypeptides. Such assays generally comprise one or more 24P4C12antibodies capable of recognizing and binding a 24P4C12 or mutant24P4C12 protein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer and other cancers expressing24P4C12 are also provided by the invention, including but limited toradioscintigraphic imaging methods using labeled 24P4C12 antibodies.Such assays may be clinically useful in the detection, monitoring, andprognosis of 24P4C12 expressing cancers, such as prostate cancer.

24P4C12 antibodies may also be used in methods for purifying 24P4C12 andmutant 24P4C12 proteins and polypeptides and for isolating 24P4C12homologues and related molecules. For example, in one embodiment, themethod of purifying a 24P4C12 protein comprises incubating a 24P4C12antibody, which has been coupled to a solid matrix, with a lysate orother solution containing 24P4C12 under conditions that permit the24P4C12 antibody to bind to 24P4C12; washing the solid matrix toeliminate impurities; and eluting the 24P4C12 from the coupled antibody.Other uses of the 24P4C12 antibodies of the invention include generatinganti-idiotypic antibodies that mimic the 24P4C12 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a 24P4C12 protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 24P4C12 mayalso be used, such as a 24P4C12 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the openreading frame amino acid sequence of FIGS. 1A–1D (SEQ ID NO: 2) may beproduced and used as an immunogen to generate appropriate antibodies. Inanother embodiment, a 24P4C12 peptide may be synthesized and used as animmunogen.

In addition, naked DNA immunization techniques known in the art may beused (with or without purified 24P4C12 protein or 24P4C12 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617–648).

The amino acid sequence of the 24P4C12 as shown in FIGS. 1A–1D (SEQ IDNO: 2) may be used to select specific regions of the 24P4C12 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the 24P4C12 amino acid sequence may be used to identifyhydrophilic regions in the 24P4C12 structure. Regions of the 24P4C12protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Methods for the generation of24P4C12 antibodies are further illustrated by way of the examplesprovided herein.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a 24P4C12 immunogen is conducted generallyby injection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

Polyclonal 24P4C12 antibodies can be prepared using conventionaltechniques known in the art. A representative protocol for thepreparation of such antibodies is described in the Examples that follow.Polyclonal antibodies can be useful for sensitive detection of multipleepitopes associated with 24P4C12.

24P4C12 monoclonal antibodies may be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody may be prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeproducing B cells, as is generally known. The immortalized cell linessecreting the desired antibodies are screened by immunoassay in whichthe antigen is the 24P4C12 protein or a 24P4C12 fragment. When theappropriate immortalized cell culture secreting the desired antibody isidentified, the cells may be expanded and antibodies produced eitherfrom in vitro cultures or from ascites fluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the 24P4C12 protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human 24P4C12 antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522–525;Riechmann et al., 1988, Nature 332: 323–327; Verhoeyen et al., 1988,Science 239: 1534–1536). See also, Carter et al., 1993, Proc. Natl.Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535–539).

Fully human 24P4C12 monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45–64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65–82). Fully human 24P4C12 monoclonal antibodies mayalso be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607–614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 24P4C12 antibodies with a 24P4C12 protein may beestablished by a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,24P4C12 proteins, peptides, 24P4C12-expressing cells or extractsthereof.

A 24P4C12 antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. A second molecule forconjugation to the 24P4C12 antibody can be selected in accordance withthe intended use. For example, for therapeutic use, the second moleculecan be a toxin or therapeutic agent. Further, bi-specific antibodiesspecific for two or more 24P4C12 epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560–2565).

H38087 Antibodies

The invention also provides antibodies, both polyclonal and monoclonal,directed against H38087. These antibodies can be modified, generated andused in manners analogous to those described above for 24P4C12antibodies. The ubiquitous expression of H38087, however, makes itlikely to be useful as a control for testing 24P4C12-specifictherapeutics, and possibly for comparison in 24P4C12 diagnosticapplications. A 24P4C12-specific therapeutic that affects H38087function could be toxic to normal cells. However, a therapeutic thatselectively affects 24P4C12, but not H38087, may be less toxic to normalcells. Therefore, H38087 proteins and antibodies can be useful aspre-clinical testing tools for therapeutic modalities directed towards24P4C12.

24P4C12 Transgenic Animals

Nucleic acids that encode 24P4C12 or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA that is integrated into the genomeof a cell from which a transgenic animal develops. In one embodiment,cDNA encoding 24P4C12 can be used to clone genomic DNA encoding 24P4C12in accordance with established techniques and the genomic sequences usedto generate transgenic animals that contain cells that express DNAencoding 24P4C12. Methods for generating transgenic animals,particularly animals such as mice or rats, have become conventional inthe art and are described, for example, in U.S. Pat. Nos. 4,736,866 and4,870,009. Typically, particular cells would be targeted for 24P4C12transgene incorporation with tissue-specific enhancers. Transgenicanimals that include a copy of a transgene encoding 24P4C12 introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of DNA encoding 24P4C12. Suchanimals can be used as tester animals for reagents thought to conferprotection from, for example, pathological conditions associated withits overexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of 24P4C12 can be used to constructa 24P4C12 “knock out” animal that has a defective or altered geneencoding 24P4C12 as a result of homologous recombination between theendogenous gene encoding 24P4C12 and altered genomic DNA encoding24P4C12 introduced into an embryonic cell of the animal. For example,cDNA encoding 24P4C12 can be used to clone genomic DNA encoding 24P4C12in accordance with established techniques. A portion of the genomic DNAencoding 24P4C12 can be deleted or replaced with another gene, such as agene encoding a selectable marker that can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113–152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the 24P4C12 polypeptide.

Likewise, H38087 transgenic animals can be prepared using nucleic acidsthat encode H38087.

Methods for the Detection of 24P4C12

Another aspect of the present invention relates to methods for detecting24P4C12 polynucleotides and 24P4C12 proteins and variants thereof, aswell as methods for identifying a cell that expresses 24P4C12. 24P4C12appears to be expressed in the LAPC xenografts that are derived fromlymph-node and bone metastasis of prostate cancer and the expressionprofile of 24P4C12 makes it a potential diagnostic marker formetastasized disease. In this context, the status of 24P4C12 geneproducts may provide information useful for predicting a variety offactors including susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. As discussed in detail below,the status of 24P4C12 gene products in patient samples may be analyzedby a variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of Northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

More particularly, the invention provides assays for the detection of24P4C12 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 24P4C12 polynucleotides include, for example, a 24P4C12gene or fragments thereof, 24P4C12 mRNA, alternative splice variant24P4C12 mRNAs, and recombinant DNA or RNA molecules containing a 24P4C12polynucleotide. A number of methods for amplifying and/or detecting thepresence of 24P4C12 polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 24P4C12 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a24P4C12 polynucleotides as sense and antisense primers to amplify24P4C12 cDNAs therein; and detecting the presence of the amplified24P4C12 cDNA. Optionally, the sequence of the amplified 24P4C12 cDNA canbe determined. In another embodiment, a method of detecting a 24P4C12gene in a biological sample comprises first isolating genomic DNA fromthe sample; amplifying the isolated genomic DNA using 24P4C12polynucleotides as sense and antisense primers to amplify the 24P4C12gene therein; and detecting the presence of the amplified 24P4C12 gene.Any number of appropriate sense and antisense probe combinations may bedesigned from the nucleotide sequences provided for the 24P4C12 FIGS.1A–1D; SEQ ID NO: 1) and used for this purpose.

The invention also provides assays for detecting the presence of a24P4C12 protein in a tissue or other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a 24P4C12 protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a 24P4C12 protein in a biological sample comprises first contactingthe sample with a 24P4C12 antibody, a 24P4C12-reactive fragment thereof,or a recombinant protein containing an antigen binding region of a24P4C12 antibody; and then detecting the binding of 24P4C12 protein inthe sample thereto.

Methods for identifying a cell that expresses 24P4C12 are also provided.In one embodiment, an assay for identifying a cell that expresses a24P4C12 gene comprises detecting the presence of 24P4C12 mRNA in thecell. Methods for the detection of particular mRNAs in cells are wellknown and include, for example, hybridization assays using complementaryDNA probes (such as in situ hybridization using labeled 24P4C12riboprobes, northern blot and related techniques) and various nucleicacid amplification assays (such as RT-PCR using complementary primersspecific for 24P4C12, and other amplification type detection methods,such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses a 24P4C12gene comprises detecting the presence of 24P4C12 protein in the cell orsecreted by the cell. Various methods for the detection of proteins arewell known in the art and may be employed for the detection of 24P4C12proteins and 24P4C12 expressing cells. 24P4C12 expression analysis mayalso be useful as a tool for identifying and evaluating agents thatmodulate 24P4C12 gene expression. For example, 24P4C12 expression issignificantly upregulated in prostate cancers, and may also be expressedin other cancers. Identification of a molecule or biological agent thatcould inhibit 24P4C12 expression or over-expression in cancer cells maybe of therapeutic value. Such an agent may be identified by using ascreen that quantifies 24P4C12 expression by RT-PCR, nucleic acidhybridization or antibody binding.

Monitoring the Status of 24P4C12 and its Products

Assays that evaluate the status of the 24P4C12 gene and 24P4C12 geneproducts in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because 24P4C12 mRNA is so highly expressed in prostate cancersrelative to normal tissues, assays that evaluate the relative levels of24P4C12 mRNA transcripts or proteins in a biological sample may be usedto diagnose a disease associated with 24P4C12 disregulation such ascancer and may provide prognostic information useful in definingappropriate therapeutic options. Similarly, assays that evaluate theintegrity 24P4C12 nucleotide and amino acid sequences in a biologicalsample, may also be used in this context.

The finding that 24P4C12 mRNA is so highly expressed in prostatecancers, and not in normal tissue, provides evidence that this gene isassociated with disregulated cell growth and therefore identifies thisgene and its products as targets that the skilled artisan can use toevaluate biological samples from individuals suspected of having adisease associated with 24P4C12 disregulation. In this context, theevaluation of the expression status of 24P4C12 gene and its products canbe used to gain information on the disease potential of a tissue sample.The terms “expression status” in this context is used to broadly referto the variety of factors involved in the expression, function andregulation of a gene and its products such as the level of mRNAexpression, the integrity of the expressed gene products (such as thenucleic and amino acid sequences) and transcriptional and translationalmodifications to these molecules.

The expression status of 24P4C12 may provide information useful forpredicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining 24P4C12 expression status and diagnosing cancers thatexpress 24P4C12, such as cancers of the prostate, breast, bladder, lung,bone, colon, pancreatic, testicular, cervical and ovarian cancers.24P4C12 expression status in patient samples may be analyzed by a numberof means well known in the art, including without limitation,immunohistochemical analysis, in situ hybridization, RT-PCR analysis onlaser capture micro-dissected samples, western blot analysis of clinicalsamples and cell lines, and tissue array analysis. Typical protocols forevaluating the expression status of the 24P4C12 gene and gene productscan be found, for example in Current Protocols In Molecular Biology,Units 2 [Northern Blotting], 4 [Southern Blotting], 15 [Immunoblotting]and 18 [PCR Analysis], Frederick M. Ausubul et al. eds., 1995.

In one aspect, the invention provides methods for monitoring 24P4C12gene products by determining the status of 24P4C12 gene productsexpressed by cells in a test tissue sample from an individual suspectedof having a disease associated with disregulated cell growth (such ashyperplasia or cancer) and then comparing the status so determined tothe status of 24P4C12 gene products in a corresponding normal sample,the presence of aberrant 24P4C12 gene products in the test samplerelative to the normal sample providing an indication of the presence ofdisregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 24P4C12 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of significant 24P4C12 expression inthese tissues may be useful to indicate the emergence, presence and/orseverity of cancer.

In a related embodiment, 24P4C12 expression status may be determined atthe protein level rather than at the nucleic acid level. For example,such a method or assay would comprise determining the level of 24P4C12protein expressed by cells in a test tissue sample and comparing thelevel so determined to the level of 24P4C12 expressed in a correspondingnormal sample. In one embodiment, the presence of 24P4C12 protein isevaluated, for example, using immunohistochemical methods. 24P4C12antibodies or binding partners capable of detecting 24P4C12 proteinexpression may be used in a variety of assay formats well known in theart for this purpose.

In other related embodiments, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdisregulated phenotype (see e.g. Marrogi et al., J. Cutan. Pathol.26(8): 369–378 (1999)). In this context, a wide variety of assays forobserving perturbations in nucleotide and amino acid sequences are wellknown in the art. For example, the size and structure of nucleic acid oramino acid sequences of 24P4C12 gene products may be observed by thenorthern, Southern, western, PCR and DNA sequencing protocols discussedherein. In addition, other methods for observing perturbations innucleotide and amino acid sequences such as single strand conformationpolymorphism analysis are well known in the art (see e.g. U.S. Pat. Nos.5,382,510 and 5,952,170).

In another related embodiment, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant change in the 24P4C12 alternative splicevariants expressed in a test cell or tissue sample relative toexpression levels in the corresponding normal cell or tissue. Themonitoring of alternative splice variants of 24P4C12 is useful becausechanges in the alternative splicing of proteins is suggested as one ofthe steps in a series of events that lead to the progression of cancers(see e.g. Carstens et al., Oncogene 15(250: 3059–3065 (1997)).

Gene amplification provides an additional method of assessing the statusof 24P4C12. Gene amplification may be measured in a sample directly, forexample, by conventional Southern blotting, northern blotting toquantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci.USA, 77:5201–5205 (1980)], dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

In addition to the tissues discussed above, peripheral blood may beconveniently assayed for the presence of cancer cells, including but notlimited to prostate cancers, using RT-PCR to detect 24P4C12 expression.The presence of RT-PCR amplifiable 24P4C12 mRNA provides an indicationof the presence of the cancer. RT-PCR detection assays for tumor cellsin peripheral blood are currently being evaluated for use in thediagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373–384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195–2000; Heston etal., 1995, Clin. Chem. 41: 1687–1688). RT-PCR assays are well known inthe art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detecting24P4C12 mRNA or 24P4C12 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 24P4C12 mRNAexpression present is proportional to the degree of susceptibility. In aspecific embodiment, the presence of 24P4C12 in prostate tissue isexamined, with the presence of 24P4C12 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In a closely related embodiment, one canevaluate the integrity 24P4C12 nucleotide and amino acid sequences in abiological sample in order to identify perturbations in the structure ofthese molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in 24P4C12 geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of 24P4C12mRNA or 24P4C12 protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of 24P4C12 mRNA or24P4C12 protein expressed in a corresponding normal tissue taken fromthe same individual or a normal tissue reference sample, wherein thedegree of 24P4C12 mRNA or 24P4C12 protein expression in the tumor samplerelative to the normal sample indicates the degree of aggressiveness. Ina specific embodiment, aggressiveness of prostate tumors is evaluated bydetermining the extent to which 24P4C12 is expressed in the tumor cells,with higher expression levels indicating more aggressive tumors. In aclosely related embodiment, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, with the presence ofone or more perturbations indicating more aggressive tumors.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 24P4C12 mRNAor 24P4C12 protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of 24P4C12 mRNA or24P4C12 protein expressed in an equivalent tissue sample taken from thesame individual at a different time, wherein the degree of 24P4C12 mRNAor 24P4C12 protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining the extent towhich 24P4C12 expression in the tumor cells alters over time, withhigher expression levels indicating a progression of the cancer. In aclosely related embodiment, one can evaluate the integrity 24P4C12nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, with the presence ofone or more perturbations indicating a progression of the cancer.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof 24P4C12 gene and 24P4C12 gene products (or perturbations in 24P4C12gene and 24P4C12 gene products) and a factor that is associated withmalignancy as a means of diagnosing and prognosticating the status of atissue sample. In this context, a wide variety of factors associatedwith malignancy may be utilized such as the expression of genesotherwise associated with malignancy (including PSA, PSCA and PSMexpression) as well as gross cytological observations (see e.g. Bockinget al., Anal Quant Cytol. 6(2):74–88 (1984); Eptsein, Hum Pathol.February 1995; 26(2):223–9 (1995); Thorson et al., Mod Pathol. June1998; 11(6):543–51; Baisden et al., Am J Surg Pathol. 23(8):918–2491999)). Methods for observing a coincidence between the expression of24P4C12 gene and 24P4C12 gene products (or perturbations in 24P4C12 geneand 24P4C12 gene products) and an additional factor that is associatedwith malignancy are useful, for example, because the presence of a setor constellation of specific factors that coincide provides informationcrucial for diagnosing and prognosticating the status of a tissuesample.

In a typical embodiment, methods for observing a coincidence between theexpression of 24P4C12 gene and 24P4C12 gene products (or perturbationsin 24P4C12 gene and 24P4C12 gene products) and a factor that isassociated with malignancy entails detecting the overexpression of24P4C12 mRNA or protein in a tissue sample, detecting the overexpressionof PSA mRNA or protein in a tissue sample, and observing a coincidenceof 24P4C12 mRNA or protein and PSA mRNA or protein overexpression. In aspecific embodiment, the expression of 24P4C12 and PSA mRNA in prostatetissue is examined. In a preferred embodiment, the coincidence of24P4C12 and PSA mRNA overexpression in the sample provides an indicationof prostate cancer, prostate cancer susceptibility or the emergence orexistence of a prostate tumor.

Methods for detecting and quantifying the expression of 24P4C12 mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of 24P4C12 mRNAinclude in situ hybridization using labeled 24P4C12 riboprobes, northernblot and related techniques using 24P4C12 polynucleotide probes, RT-PCRanalysis using primers specific for 24P4C12, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR may beused to detect and quantify 24P4C12 mRNA expression as described in theExamples that follow. Any number of primers capable of amplifying24P4C12 may be used for this purpose, including but not limited to thevarious primer sets specifically described herein. Standard methods forthe detection and quantification of protein may be used for thispurpose. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 24P4C12 protein may be used inan immunohistochemical assay of biopsied tissue.

Identifying Molecules that Interact with 24P4C12

The 24P4C12 protein sequences disclosed herein allow the skilled artisanto identify molecules that interact with them via any one of a varietyof art accepted protocols. For example one can utilize one of thevariety of so-called interaction trap systems (also referred to as the“two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively, one can identify molecules that interact with 24P4C12protein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as 24P4C12 areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest. Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with 24P4C12 protein sequencesare disclosed for example in U.S. Pat. Nos. 5,723,286 and 5,733,731.

Alternatively, cell lines expressing 24P4C12 can be used to identifyprotein-protein interactions mediated by 24P4C12. This possibility canbe examined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646–51).Typically 24P4C12 protein can be immunoprecipitated from 24P4C12expressing prostate cancer cell lines using anti-24P4C12 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express 24P4C12 (using, e.g., vectors mentioned above).The immuno-precipitated complex can be examined for protein associationby procedures such as western blotting, ³⁵S-methionine labeling ofproteins, protein microsequencing, silver staining and two dimensionalgel electrophoresis.

Related embodiments of such screening assays include methods foridentifying small molecules that interact with 24P4C12. Typical methodsare discussed for example in U.S. Pat. No. 5,928,868 and include methodsfor forming hybrid ligands in which at least one ligand is a smallmolecule. In an illustrative embodiment, the hybrid ligand is introducedinto cells that in turn contain a first and a second expression vector.Each expression vector includes DNA for expressing a hybrid protein thatencodes a target protein linked to a coding sequence for atranscriptional module. Each cell further contains a reporter gene, theexpression of which is conditioned on the proximity of the first andsecond hybrid proteins to each other, an event that occurs only if thehybrid ligand binds to target sites on both hybrid proteins. Those cellsthat express the reporter gene are selected and the unknown smallmolecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with a 24P4C12 amino acid sequence shownin FIGS. 1A–1D, comprising the steps of contacting a population ofmolecules with the 24P4C12 amino acid sequence, allowing the populationof molecules and the 24P4C12 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 24P4C12 amino acid sequence and thenseparating molecules that do not interact with the 24P4C12 amino acidsequence from molecules that do interact with the 24P4C12 amino acidsequence. In a specific embodiment, the method further includespurifying a molecule that interacts with the 24P4C12 amino acidsequence. In a preferred embodiment, the 24P4C12 amino acid sequence iscontacted with a library of peptides.

Therapeutic Methods and Compositions

The identification of 24P4C12 as a prostate tumor-associated protein,opens a number of therapeutic approaches to the treatment of suchcancers. As discussed above, it is possible that 24P4C12 functions as areceptor involved in activating or modulating proliferation signals, andthat it presents epitopes at the cell surface that can be targeted fortherapy.

The expression profile of 24P4C12 is reminiscent of the MAGEs, PSA andPMSA, which are tissue-specific genes that are up-regulated in melanomasand other cancers (Van den Eynde and Boon, Int J Clin Lab Res. 27:81–86,1997). Due to their tissue-specific expression and high expressionlevels in cancer, these molecules are currently being investigated astargets for cancer vaccines (Durrant, Anticancer Drugs 8:727–733, 1997;Reynolds et al., Int J Cancer 72:972–976, 1997). The expression patternof 24P4C12 provides evidence that it is likewise a potential target fora cancer vaccine approach to prostate cancer and other cancers, as itsexpression is limited in normal tissues. Its structural features as apotential receptor also provides evidence that 24P4C12 may be a smallmolecule target, as well as a target for antibody-based therapeuticstrategies.

Accordingly, therapeutic approaches targeting extracellular portions of24P4C12, or aimed at inhibiting the activity of the 24P4C12 protein areexpected to be useful for patients suffering from prostate cancer,testicular cancer, and other cancers expressing 24P4C12. The therapeuticapproaches aimed at inhibiting the activity of the 24P4C12 proteingenerally fall into two classes. One class comprises various methods forinhibiting the binding or association of the 24P4C12 protein with itsbinding partner or with other proteins. Another class comprises avariety of methods for inhibiting the transcription of the 24P4C12 geneor translation of 24P4C12 mRNA.

24P4C12 as a Cell Surface Target for Antibody-Based Therapy

The structural features of 24P4C12 indicate that this molecule is likelya cell surface antigen, providing an attractive target forantibody-based therapeutic strategies. Because 24P4C12 is over-expressedon cancer cells relative to normal cells, systemic administration of24P4C12-immunoreactive compositions would be expected to exhibitrelatively good sensitivity with minimal toxic, non-specific and/ornon-target effects caused by binding of the immunotherapeutic moleculeto non-target organs and tissues. Antibodies specifically reactive withextracellular domains of 24P4C12 can be useful to treat24P4C12-expressing cancers systemically, either as conjugates with atoxin or therapeutic agent, or as naked antibodies capable of inhibitingcell proliferation or function.

24P4C12 antibodies can be introduced into a patient such that theantibody binds to 24P4C12 on the cancer cells and mediates thedestruction of the cells and the tumor and/or inhibits the growth of thecells or the tumor. Mechanisms by which such antibodies exert atherapeutic effect may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, modulating the physiologicalfunction of 24P4C12, inhibiting ligand binding or signal transductionpathways, modulating tumor cell differentiation, altering tumorangiogenesis factor profiles, and/or by inducing apoptosis. 24P4C12antibodies can be conjugated to toxic or therapeutic agents and used todeliver the toxic or therapeutic agent directly to 24P4C12-bearing tumorcells. Examples of toxic agents include, but are not limited to,calchemicin, maytansinoids, radioisotopes such as ¹³¹I, ytrium, andbismuth.

Cancer immunotherapy using anti-24P4C12 antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133–138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179–3186;Tsunenari et al., 1997, Blood 90:2437–2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771–2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93–101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581–589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160–6166); Velders et al., 1995, Cancer Res.55:4398–4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117–127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of ¹³¹I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp., naked antibody;Coulter Pharmaceuticals, Palo Alto, Calif., ¹³¹I-anti-CD20 conjugate),while others involve co-administration of antibodies and othertherapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel(Genentech, Inc.). For treatment of prostate cancer, for example,24P4C12 antibodies can be administered in conjunction with radiation,chemotherapy or hormone ablation.

Although 24P4C12 antibody therapy may be useful for all stages ofcancer, antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It may be desirable for some cancer patients to be evaluated for thepresence and level of 24P4C12 expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative 24P4C12imaging, or other techniques capable of reliably indicating the presenceand degree of 24P4C12 expression. Immunohistochemical analysis of tumorbiopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-24P4C12 monoclonal antibodies useful in treating prostate and othercancers include those that are capable of initiating a potent immuneresponse against the tumor and those that are capable of directcytotoxicity. In this regard, anti-24P4C12 monoclonal antibodies (mAbs)may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-24P4C12 mAbs that exert a direct biological effect ontumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-24P4C12 mAb exertsan anti-tumor effect may be evaluated using any number of in vitroassays designed to determine ADCC, ADMMC, complement-mediated celllysis, and so forth, as is generally known in the art.

The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 24P4C12antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-24P4C12 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination mayexhibit synergistic therapeutic effects. In addition, the administrationof anti-24P4C12 mAbs may be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-24P4C12 mAbs may be administered in their “naked” or unconjugatedform, or may have therapeutic agents conjugated to them.

The anti-24P4C12 antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumor, intradermal,and the like. Treatment will generally involve the repeatedadministration of the anti-24P4C12 antibody preparation via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. Doses in the range of 10–500 mg mAb per week may be effectiveand well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-24P4C12 mAb preparation may represent an acceptabledosing regimen. Preferably, the initial loading dose is administered asa 90 minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of 24P4C12expression in the patient, the extent of circulating shed 24P4C12antigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed 24P4C12 antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Inhibition of 24P4C12 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 24P4C12 to its binding partner or ligand, or itsassociation with other protein(s) as well as methods for inhibiting24P4C12 function.

Inhibition of 24P4C12 with Intracellular Antibodies

In one approach, recombinant vectors encoding single chain antibodiesthat specifically bind to 24P4C12 may be introduced into 24P4C12expressing cells via gene transfer technologies, wherein the encodedsingle chain anti-24P4C12 antibody is expressed intracellularly, bindsto 24P4C12 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137–3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931–23936; Deshane et al., 1994, Gene Ther.1: 332–337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In order to specifically direct the expression of such intrabodies toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of 24P4C12 with Recombinant Proteins

In another approach, recombinant molecules that are capable of bindingto 24P4C12 thereby preventing 24P4C12 from accessing/binding to itsbinding partner(s) or associating with other protein(s) are used toinhibit 24P4C12 function. Such recombinant molecules may, for example,contain the reactive part(s) of a 24P4C12 specific antibody molecule. Ina particular embodiment, the 24P4C12 binding domain of a 24P4C12 bindingpartner may be engineered into a dimeric fusion protein comprising two24P4C12 ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion may contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins may be administered in soluble formto patients suffering from a cancer associated with the expression of24P4C12, where the dimeric fusion protein specifically binds to 24P4C12thereby blocking 24P4C12 interaction with a binding partner. Suchdimeric fusion proteins may be further combined into multimeric proteinsusing known antibody linking technologies.

Inhibition of 24P4C12 Transcription or Translation

Within another class of therapeutic approaches, the invention providesvarious methods and compositions for inhibiting the transcription of the24P4C12 gene. Similarly, the invention also provides methods andcompositions for inhibiting the translation of 24P4C12 mRNA intoprotein.

In one approach, a method of inhibiting the transcription of the 24P4C12gene comprises contacting the 24P4C12 gene with a 24P4C12 antisensepolynucleotide. In another approach, a method of inhibiting 24P4C12 mRNAtranslation comprises contacting the 24P4C12 mRNA with an antisensepolynucleotide. In another approach, a 24P4C12 specific ribozyme may beused to cleave the 24P4C12 message, thereby inhibiting translation. Suchantisense and ribozyme based methods may also be directed to theregulatory regions of the 24P4C12 gene, such as the 24P4C12 promoterand/or enhancer elements. Similarly, proteins capable of inhibiting a24P4C12 gene transcription factor may be used to inhibit 24P4C12 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 24P4C12 throughinterfering with 24P4C12 transcriptional activation may also be usefulfor the treatment of cancers expressing 24P4C12. Similarly, factors thatare capable of interfering with 24P4C12 processing may be useful for thetreatment of cancers expressing 24P4C12. Cancer treatment methodsutilizing such factors are also within the scope of the invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizing 24P4C12(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 24P4C12 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 24P4C12 antisensepolynucleotides, ribozymes, factors capable of interfering with 24P4C12transcription, and so forth, may be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches may be combined with any one of a widevariety of chemotherapy or radiation therapy regimens. These therapeuticapproaches may also enable the use of reduced dosages of chemotherapyand/or less frequent administration, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 24P4C12 to a bindingpartner, etc.

In vivo, the effect of a 24P4C12 therapeutic composition may beevaluated in a suitable animal model. For example, xenogenic prostatecancer models wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice, are appropriate in relation to prostate cancer andhave been described (Klein et al., 1997, Nature Medicine 3: 402–408).For example, PCT Patent Application WO98/16628, Sawyers et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isnon-reactive with the patient's immune system. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like (see, generally, Remington's Pharmaceutical Sciences16^(th) Edition, A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile sodium chloride for injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

The invention further provides cancer vaccines comprising a 24P4C12protein or fragment thereof, as well as DNA based vaccines. Preferably,the vaccine comprises an immunogenic portion of a 24P4C12 protein orpolypeptide. In view of the tissue-restricted expression of 24P4C12,24P4C12 cancer vaccines are expected to be effective at specificallypreventing and/or treating 24P4C12 expressing cancers without creatingnon-specific effects on non-target tissues. The use of a tumor antigenin a vaccine for generating humoral and cell-mediated immunity for usein anti-cancer therapy is well known in the art and has been employed inprostate cancer using human PSMA and rodent PAP immunogens (Hodge etal., 1995, Int. J. Cancer 63: 231–237; Fong et al., 1997, J. Immunol.159: 3113–3117). Such methods can be readily practiced by employing a24P4C12 protein, or fragment thereof, or a 24P4C12-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the 24P4C12 immunogen.

For example, viral gene delivery systems may be used to deliver a24P4C12-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658–663).Non-viral delivery systems may also be employed by using naked DNAencoding a 24P4C12 protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human 24P4C12 cDNA may be employed. Inanother embodiment, 24P4C12 nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a 24P4C12 protein that are capable ofoptimally binding to specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present 24P4C12 antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65–69; Murphy et al.,1996, Prostate 29: 371–380). Dendritic cells can be used to present24P4C12 peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed with24P4C12 peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete 24P4C12protein. Yet another embodiment involves engineering the overexpressionof the 24P4C12 gene in dendritic cells using various implementingvectors known in the art, such as adenovirus (Arthur et al., 1997,Cancer Gene Ther. 4: 17–25), retrovirus (Henderson et al., 1996, CancerRes. 56: 3763–3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57: 2865–2869), andtumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177–1182). Cells expressing 24P4C12 may also be engineered to expressimmune modulators, such as GM-CSF, and used as immunizing agents.

Anti-idiotypic anti-24P4C12 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 24P4C12 protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-24P4C12 antibodies that mimic an epitope ona 24P4C12 protein (see, for example, Wagner et al., 1997, Hybridoma 16:33–40; Foon et al., 1995, J Clin Invest 96: 334–342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65–76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing 24P4C12. Constructs comprising DNA encoding a24P4C12 protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded 24P4C12 protein/immunogen. Expression of the 24P4C12 proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate cancers. Variousprophylactic and therapeutic genetic immunization techniques known inthe art may be used (for review, see information and referencespublished at Internet address www.genweb.com).

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a 24P4C12(and/or H38087) protein or a 24P4C12 (and/or H38087) gene or message,respectively. Where the kit utilizes nucleic acid hybridization todetect the target nucleic acid, the kit may also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence and/or a container comprising a reporter-means, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, florescent, or radioisotopelabel.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The 24P4C12 cDNA was deposited under the terms of the Budapest Treatywith the American Type Culture Collection (ATCC; 10801 University Blvd.,Manassas, Va. 20110-2209 USA) as plasmids p24P4C12-GTE9 andp24P4C12-GTE5 on Feb. 2 and 26, 1999, respectively, and have beenaccorded ATCC Designation Numbers 207084 and 207129.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 24P4C12 Gene

Materials and Methods

LAPC Xenografts

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3:402–408; Craftet al., 1999, Cancer Res. 59:5030–5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and AI, respectively) weregrown in intact male SCID mice or in castrated males, respectively, andwere passaged as small tissue chunks in recipient males. LAPC-4 AIxenografts were derived from LAPC-4 AD tumors. To generate the AIxenografts, male mice bearing LAPC AD tumors were castrated andmaintained for 2–3 months. After the LAPC tumors re-grew, the tumorswere harvested and passaged in castrated males or in female SCID mice.

Cell Lines

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 5% fetal calf serum.

RNA Isolation

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 34)Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NOS:35, 36, respectively) 3′GGCCCGTCCTAG5′ Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NOS: 37, 38,respectively) 3′CGGCTCCTAG5′ PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′(SEQ ID NO: 39) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ IDNO: 40) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 41)Suppression Subtractive Hybridization

Suppression subtractive hybridization (SSH) was used to identify cDNAscorresponding to 24P4C12s that may be differentially expressed inprostate cancer. The SSH reaction utilized cDNA from two differentprostate tissue sources, subtracting BPH (benign prostatic hyperplasia)cDNA from LAPC-4 AD cDNA. The LAPC-4 AD cDNA was used as the source ofthe “tester”, while the BPH cDNA was used as the source of the “driver”.

Double stranded cDNAs corresponding to tester and driver cDNAs weresynthesized from 2 μg of poly(A)+ RNA isolated from the relevantxenograft tissue, as described above, using CLONTECH's PCR-Select cDNASubtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- andsecond-strand synthesis were carried out as described in the Kit's usermanual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1).The resulting cDNA was digested with Dpn II for 3 hrs. at 37° C.Digested cDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant xenograft source (see above) with a mix ofdigested cDNAs derived from human benign prostatic hyperplasia (BPH),the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA fromthe relevant xenograft source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10–12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12–18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:42) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 43) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1× PCR buffer(Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1XKlentaq DNA polymerase (Clontech). Five μl of the PCR reaction wasremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: initial denaturation was at 94° C. for15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for2 min. After agarose gel electrophoresis, the band intensities of the283 bp β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization were required to achieveequal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 24P4C12 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs, which were designed withthe assistance of (MIT; for details, see, www.genome.wi.mit.edu):

5′-agatgaggaggaggacaaaggtg-3′ (SEQ ID NO:44)5′-actgctgggaggagtaccgagtg-3′ (SEQ ID NO:45)

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results

The SSH experiments described in the Materials and Methods, supra, ledto the isolation of numerous candidate gene fragment clones (SSHclones). All candidate clones were sequenced and subjected to homologyanalysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or northern analysis.

One of the SHH clones, comprising about 160 bp, encodes a putative openreading frame (ORF) of 53 amino acids and exhibits significant homologyto an EST derived from a colon tumor library (FIG. 1E; SEQ ID NOS: 3,4). This SSH clone, designated 24P4C12, was used to design primers forRT-PCR expression analysis of the 24P4C12 gene in various tissues.RT-PCR analysis showed that 24P4C12 is expressed in all the LAPCxenografts and normal prostate at approximately equal levels (FIG. 2A).RT-PCR analysis of first strand cDNA derived from 16 normal tissuesshowed expression in colon, prostate, kidney and lung after 25 cycles ofamplification (FIGS. 2B and 2C). Northern blot analysis using the24P4C12 SSH fragment as probe shows the highest expression of anapproximately 3 kb 24P4C12 transcript in LAPC-9AD, followed by LAPC-4 AD(FIGS. 3A–3C).

Example 2 Cloning of Full Length 24P4C12 cDNAs

Full length cDNAs encoding the 24P4C12 gene were isolated from a normalprostate library and sequenced. Two of the isolated clones, designated24P4C12-GTE9 (containing most of the coding region of the 24P4C12 gene)and 24P4C12-GTE5 (containing the entire coding region of the 24P4C12gene), were deposited as plasmids p24P4C12-GTE9 and p24P4C12-GTE5 withthe ATCC (Manassas, Va.) on Feb. 2 and 26, 1999, respectively, and havebeen accorded ATCC Designation Numbers 207084 and 207129, respectively.These two clones, as well as another clone encoding most of the 24P4C12coding region, 24P4C12-GTE4, had overlapping nucleotide sequences whichwere combined to generate the complete coding and partial non-codingsequence of the 24P4C12 gene as shown in FIGS. 1A–1D (SEQ ID NO: 1).

The 2587 bp 24P4C12 cDNA sequence shown in FIGS. 1A–1D (SEQ ID NO: 1)encodes an ORF of 710 amino acids with significant homology to the mousegene NG22 and the C. elegans gene CEESB82F. An amino acid sequencealignment of the human 24P4C12 protein encoded by the cDNA of FIGS.1A–1D (SEQ ID NO: 2) and the murine NG22 gene products is shown in FIGS.4A–4B.

NG22 was recently identified as one of many ORFs within a genomic BACclone that encompasses the MHC class III in the mouse genome. Both NG22and CEESB82F appear to be genes that contain 12 transmembrane domains.The 24P4C12 cDNA sequence shown in FIGS. 1A–1D (SEQ ID NO: 1) contains13 potential transmembrane domains. 12-transmembrane transporterproteins are known (Kitty and Amara, 1992, Curr. Opin. Biotechnology 3:675–682). Due to the putative secondary structure of 24P4C12, it ispossible that the 24P4C12 protein functions as a cell surface membranepump or transporter.

Example 3 24P4C12 Gene Expression Analysis

24P4C12 mRNA expression in normal human tissues was analyzed by northernblotting of multiple tissue blots (Clontech; Palo Alto, Calif.),comprising a total of 16 different normal human tissues, using labeled24P4C12 SSH fragment (Example 1) as a probe. RNA samples werequantitatively normalized with a β-actin probe. Northern blot analysisshowed expression primarily in prostate and colon, with lower expressiondetected in kidney, and significantly lower expression detected inpancreas, lung and placenta.

To analyze 24P4C12 expression in cancer tissues, northern blotting wasperformed on RNA derived from the LAPC xenografts, and several prostateand non-prostate cancer cell lines. The results show high expressionlevels of 24P4C12 in LAPC-4 AD, LAPC-4 AI, LAPC-9 AD, LNCaP and LAPC-4cell line (FIG. 3, FIG. 5). Very high levels are detected in LAPC-3 AI(FIG. 5). Lower levels are detected in LAPC-9 AI. More detailed analysisof the xenografts shows that 24P4C12 is highly expressed in thexenografts even when grown within the tibia of mice (FIG. 5). Northernanalysis also shows that 24P4C12 is expressed in the normal prostate andprostate tumor tissues derived from prostate cancer patients (FIG. 6A).These results suggest that 24P4C12 is a prostate gene that is highlyexpressed in prostate cancer and may have a utility as a drug orantibody target in prostate cancer.

Example 4 Generation of 24P4C12 Polyclonal Antibodies

To generate polyclonal sera to 24P4C12, a peptide was synthesizedcorresponding to amino acids 1–14 (MGGKQRDEDDEAYG; SEQ ID NO: 71) of the24P4C12 protein sequence. This peptide was coupled to Keyhole limpethemacyanin (KLH) and was used to immunize a rabbit as follows. Therabbit was initially immunized with 200 μg of peptide-KLH mixed incomplete Freund's adjuvant. The rabbit was then injected every two weekswith 200 μg of peptide-KLH in incomplete Freund's adjuvant. Bleeds weretaken approximately 7–10 days following each immunization. ELISA andWestern blotting analyses were used to determine titer and specificityof the rabbit serum to the immunizing peptide and 24P4C12 proteinrespectively. Affinity purified anti-24P4C12 polyclonal antibodies wereprepared by passage of crude serum from immunized rabbit over anaffinity matrix comprised of 24P4C12 peptide covalently coupled toAffigel 15 (BioRad). After extensive washing of the matrix with PBS,antibodies specific to 24P4C12 peptide were eluted with low pH glycinebuffer (0.1M, pH 2.5) and dialyzed against PBS.

Western blot analysis was then performed with anti-24P4C12 pAb of 293Tcells transiently transfected with 24P4C12 cDNA either in the CMV-drivenPCDNA3.1 Myc-His (Invitrogen) or retroviral pSR-alpha expressionvectors. 293T cells were transiently transfected with 10 μg of eitherempty vector, or with the 24P4C12 cDNA in pCDNA 3.1 CMV-driven MYC-His(Invitrogen) or pSR-alpha retroviral expression vectors using the CaPO4method. Forty hours following transfection cells were harvested andlysed in 2× SDS-PAGE sample buffer. Cell lysates in sample buffer werethen subjected to either mild heat denaturation (70° C.) or strong heatdenaturation (100° C.), separated on a 10% SDS-PAGE gel and transferredto nitrocellulose. Membranes were then subjected to Western analysiswith 2 μg/ml of an affinity purified rabbit anti-peptide pAb raised toamino acids 1–14 (MGGKQRDEDDEAYG; SEQ ID NO: 71) of 24P4C12.Anti-24P4C12 immunoreactive bands were visualized by incubation withanti-rabbit-HRP conjugated secondary antibody and enhancedchemiluminescence detection.

Results of the western blot analysis show specific recognition of a 90kD band and of a high molecular smear in transfected cells but not incells transfected with empty vector (FIG. 10A, FIG. 10B). The calculatedmolecular weight of 24P4C12 from the amino acid sequence is 79.2 kD. Theappearance of a 90 kD band in western analysis of cell lysates suggeststhat 24P4C12 protein contains post-translational modifications. Indeed,there are multiple potential N-linked glycosylation sites predicted fromthe amino acid sequence. The ratio of the high molecular smear isenhanced by high heat (100° C.) denaturation compared to mild heat (70°C.) denaturation which suggests aggregation of this 12 transmembraneprotein upon heat-induced exposure of hydrophobic sequences. Multiplelower molecular weight bands are also detected in cells transfected withthe highly expressed pcDNA 3.1 vector that may represent degradationproducts.

Example 5 Production of Recombinant 24P4C12 in a Mammalian System

To express recombinant 24P4C12, the full length 24P4C12 cDNA can becloned into an expression vector that provides a 6His tag at thecarboxyl-terminus (pCDNA 3.1 myc-his, InVitrogen). The constructs can betransfected into 293T cells. Transfected 293T cell lysates can be probedwith the anti-24P4C12 polyclonal serum described in Example 4 above in aWestern blot.

The 24P4C12 genes can also be subcloned into the retroviral expressionvector pSRαMSVtkneo and used to establish 24P4C12 expressing cell linesas follows. The 24P4C12 coding sequence (from translation initiation ATGto the termination codons) is amplified by PCR using ds cDNA templatefrom 24P4C12 cDNA. The PCR product is subcloned into pSRαMSVtkneo viathe EcoR1(blunt-ended) and Xba 1 restriction sites on the vector andtransformed into DH5α competent cells. Colonies are picked to screen forclones with unique internal restriction sites on the cDNA. The positiveclone is confirmed by sequencing of the cDNA insert. Retroviruses maythereafter be used for infection and generation of various cell linesusing, for example, NIH 3T3, PC3, and LnCap cells.

Example 6 Production of Recombinant 24P4C12 in a Baculovirus System

To generate a recombinant 24P4C12 protein in a baculovirus expressionsystem, the 24P4C12 cDNA is cloned into the baculovirus transfer vectorpBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminusSpecifically, pBlueBac-24P4C12 is co-transfected with helper plasmidpBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cellsto generate recombinant baculovirus (see Invitrogen instruction manualfor details). Baculovirus is then collected from cell supernatant andpurified by plaque assay.

Recombinant 24P4C12 protein is then generated by infection of HighFiveinsect cells (4nVitrogen) with the purified baculovirus. Recombinant24P4C12 protein may be detected using anti-24P4C12 antibody. 24P4C12protein may be purified and used in various cell based assays or asimmunogen to generate polyclonal and monoclonal antibodies specific for24P4C12.

Example 7 Identification & Cloning of H38087, Family Member of 24P4C12

H38087 was identified as a family member of 24P4C12 by searching thedBEST database with the 24P4C12 amino acid sequence using the tblastntool in NCBI. ESTs that encode protein fragments of homologous proteinswere identified. One of these, H38087, was cloned from a testis library.The cDNA (clone GTB6) is 2738 bp in size and encodes a 704 amino acidprotein with 11 putative transmembrane domains (FIGS. 7A–7D; SEQ ID NOS:6, 7). The 58 base pairs of 5′ untranslated region are very GC rich(87%), indicating that this gene may contain translational regulatoryelements (FIGS. 7A–7D). The amino acid sequence of 24P4C12 (SEQ ID NO:2) and H38087 (SEQ ID NO: 7) are 44% identical and 56% homologous overthe entire sequence (FIG. 8). Expression analysis shows that H38087 isubiquitously expressed (FIG. 9). Highest expression levels are detectedin testis. Expression is also seen in all the LAPC xenografts. SinceH38087 is ubiquitously expressed, it could serve as a control fortesting 24P4C12-specific therapeutics. A 24P4C12-specific therapeuticthat affects H38087 function could be toxic to normal cells. However, atherapeutic that selectively affects 24P4C12, but not H38087, may beless toxic to normal cells. Therefore, H38087 protein is useful as apre-clinical testing tool for therapeutic modalities directed towards24P4C12.

Example 8 Identification of Potential Signal Transduction Pathways

To determine whether 24P4C12 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressing24P4C12. These transcriptional reporters contain consensus binding sitesfor known transcription factors that lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof their associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

-   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress-   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation-   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress-   4. ARE-luc, androgen receptor; steroids/MAPK;    growth/differentiation/apoptosis-   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis-   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

24P4C12-mediated effects may be assayed in cells showing mRNAexpression. Luciferase reporter plasmids may be introduced by lipidmediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cells extracts with luciferin substrate and luminescenceof the reaction is monitored in a luminometer.

Example 9 Generation of 24P4C12 Monoclonal Antibodies

In order to generate 24P4C12 monoclonal antibodies, aglutathione-S-transferase (GST) fusion protein encompassing a 24P4C12protein is synthesized and used as immunogen. Alternatively, 24P4C12 canbe conveniently expressed in 293T cells transfected with a CMV-drivenexpression vector encoding 24P4C12 with a C-terminal 6× His and MYC tag(pcDNA3.1/mycHIS, Invitrogen). HIS-tagged 24P4C12 expressed in cells canbe purified using a nickel column using standard techniques.

Balb C mice are initially immunized intraperitoneally with 200 μg of theGST-24P4C12 fusion protein mixed in complete Freund's adjuvant. Mice aresubsequently immunized every 2 weeks with 75 μg of GST-24P4C12 proteinmixed in Freund's incomplete adjuvant for a total of 3 immunizations.Reactivity of serum from immunized mice to full length 24P4C12 proteinis monitored by ELISA using a partially purified preparation ofHIS-tagged 24P4C12 protein expressed from 293T cells (Example 5). Miceshowing the strongest reactivity are rested for 3 weeks and given afinal injection of fusion protein in PBS and then sacrificed 4 dayslater. The spleens of the sacrificed mice are then harvested and fusedto SPO/2 myeloma cells using standard procedures (Harlow and Lane,1988). Supernatants from growth wells following HAT selection arescreened by ELISA and Western blot to identify 24P4C12 specific antibodyproducing clones.

The binding affinity of a 24P4C12 monoclonal antibody may be determinedusing standard technology. Affinity measurements quantify the strengthof antibody to epitope binding and may be used to help define which24P4C12 monoclonal antibodies are preferred for diagnostic ortherapeutic use. The BIAcore system (Uppsala, Sweden) is a preferredmethod for determining binding affinity. The BIAcore system uses surfaceplasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Mortonand Myszka, 1998, Methods in Enzymology 295: 268) to monitorbiomolecular interactions in real time. BIAcore analysis convenientlygenerates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants.

Example 10 In Vitro Assays of 24P4C12 Function

The expression of 24P4C12 in prostate and other cancers providesevidence that this gene has a functional role in tumor progressionand/or tumor initiation. It is possible that 24P4C12 functions as areceptor involved in activating proliferation signals. 24P4C12 functioncan be assessed in mammalian cells using in vitro approaches. Formammalian expression, 24P4C12 can be cloned into a number of appropriatevectors, including pcDNA 3.1 myc-His-tag (Example 5) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using suchexpression vectors, 24P4C12 can be expressed in several cell lines,including PC-3, NIH 3T3, LNCAP and 293T. Expression of 24P4C12 can bemonitored using anti-24P4C12 antibodies and northern blot analysis (seeExamples 4 and 9).

Mammalian cell lines expressing 24P4C12 can be tested in several invitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS;Welch et al., Int. J. Cancer 43: 449–457). 24P4C12 cell phenotype iscompared to the phenotype of cells that lack expression of 24P4C12.

Cell lines expressing 24P4C12 can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and 24P4C12 overexpressing PC3, NIH 3T3 andLNCaP cells. To determine whether 24P4C12-expressing cells havechemoattractant properties, indicator cells are monitored for passagethrough the porous membrane toward a gradient of 24P4C12 conditionedmedia compared to control media. This assay may also be used to qualifyand quantify specific neutralization of the 24P4C12 induced effect bycandidate cancer therapeutic compositions.

The function of 24P4C12 can be evaluated using anti-sense RNA technologycoupled to the various functional assays described above, e.g. growth,invasion and migration. Anti-sense RNA oligonucleotides can beintroduced into 24P4C12 expressing cells, thereby preventing theexpression of 24P4C12. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of 24P4C12 expression can be evaluated.

Example 11 In Vivo Assay for 24P4C12 Tumor Growth Promotion

The effect of the 24P4C12 protein on tumor cell growth may be evaluatedin vivo by gene overexpression in tumor-bearing mice. For example, SCIDmice can be injected subcutaneously on each flank with 1×10⁶ of eitherPC3, TSUPR1, or DU145 cells containing tkNeo empty vector or 24P4C12. Atleast two strategies may be used: (1) Constitutive 24P4C12 expressionunder regulation of a promoter such as a constitutive promoter obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, avian sarcoma virus, cytomegalovirus, aretrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, provided such promoters are compatible with thehost cell systems, and (2) Regulated expression under control of aninducible vector system, such as ecdysone, tet, etc., provided suchpromoters are compatible with the host cell systems. Tumor volume isthen monitored at the appearance of palpable tumors and followed overtime to determine if 24P4C12 expressing cells grow at a faster rate andwhether tumors produced by 24P4C12-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).Additionally, mice may be implanted with 1×10⁵ of the same cellsorthotopically to determine if 24P4C12 has an effect on local growth inthe prostate or on the ability of the cells to metastasize, specificallyto lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 24P4C12 inhibitory effect ofcandidate therapeutic compositions, such as for example, 24P4C12intrabodies, 24P4C12 antisense molecules and ribozymes.

Example 12 Western Analysis of 24P4C12 Expression in SubcellularFractions

Sequence analysis of 24P4C12 revealed the presence of a transmembranedomain. The cellular location of 24P4C12 can be assessed usingsubcellular fractionation techniques widely used in cellular biology(Storrie B, et al. Methods Enzymol. 1990;182:203–25). Prostate celllines can be separated into nuclear, cytosolic and membrane fractions.The expression of 24P4C12 in the different fractions can be tested usingwestern blotting techniques.

Alternatively, to determine the subcellular localization of 24P4C12,293T cells can be transfected with an expression vector encodingHIS-tagged 24P4C12 (PCDNA 3.1 MYC/HIS, Invitrogen). The transfectedcells can be harvested and subjected to a differential subcellularfractionation protocol as previously described (Pemberton, P. A. et al,1997, J of Histochemistry and Cytochemistry, 45:1697–1706.) Thisprotocol separates the cell into fractions enriched for nuclei, heavymembranes (lysosomes, peroxisomes, and mitochondria), light membranes(plasma membrane and endoplasmic reticulum), and soluble proteins.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention. These modifications and other embodimentsinclude, but are not limited to, adapting the various methods, assays,molecules and strategies disclosed herein in connection with 24P4C12 foruse with H38087.

1. An isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or a variant thereof at least 90% identical to the amino acid sequence of SEQ ID NO:2 over its entire length, wherein amino acid substitutions are conservative amino acid substitutions, and which polypeptide raises antibodies that bind the extracellular region of the protein consisting of the amino acid sequence of SEQ ID NO:2.
 2. The polypeptide of claim 1 further comprising a covalently bound moiety.
 3. The polypeptide of claim 2 wherein said moiety is a polyoxyalkylene.
 4. The polypeptide of claim 2 wherein said moiety comprises at least one glycose.
 5. An immunogenic composition which comprises the polypeptide of claim 1 in admixture with at least one pharmaceutically acceptable excipient.
 6. A method to produce antibodies in a subject which method comprises administering to said subject an amount of the polypeptide of claim 1 sufficient to induce said immune response. 